Biodegradable poly(beta-amino esters) and uses thereof

ABSTRACT

Poly(β-amino esters) prepared from the conjugate addition of bis(secondary amines) or primary amines to a bis(acrylate ester) are described. Methods of preparing these polymers from commercially available starting materials are also provided. These tertiary amine-containing polymers are preferably biodegradable and biocompatible and may be used in a variety of drug delivery systems. Given the poly(amine) nature of these polymers, they are particularly suited for the delivery of polynucleotides. Nanoparticles containing polymer/polynucleotide complexes have been prepared. The inventive polymers may also be used to encapsulate other agents to be delivered. They are particularly useful in delivering labile agents given their ability to buffer the pH of their surroundings.

RELATED APPLICATIONS

The present application is a divisional of and claims priority under 35U.S.C. §120 to U.S. patent application, U.S. Ser. No. 12/507,999, filedJul. 23, 2009, which is a continuation of and claims priority under 35U.S.C. §120 to U.S. patent application, U.S. Ser. No. 11/099,886, filedApr. 6, 2005, which is a divisional of and claims priority under 35U.S.C. §120 to U.S. patent application, U.S. Ser. No. 09/969,431, filedOct. 2, 2001, now issued as U.S. Pat. No. 6,998,115, which claimspriority under 35 U.S.C. §119(e) to U.S. provisional applications, U.S.Ser. No. 60/305,337, filed Jul. 13, 2001, and U.S. Ser. No. 60/239,330,filed Oct. 10, 2000; each of which is incorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with Government support under Grant No.ECC9843342, awarded by the National Science Foundation; under Grant No.GM26698 and Grant No. 1 F32 GM20227-01, awarded by the NationalInstitutes of Health; and under DAMD 17-99-2-9-001, awarded by theDepartment of the Army. The Government has certain rights in thisinvention.

BACKGROUND OF THE INVENTION

The treatment of human diseases through the application ofnucleotide-based drugs such as DNA and RNA has the potential torevolutionize the medical field (Anderson Nature 392(Suppl.):25-30,1996; Friedman Nature Med. 2:144-147, 1996; Crystal Science 270:404-410,1995; Mulligan Science 260:926-932, 1993; each of which is incorporatedherein by reference). Thus far, the use of modified viruses as genetransfer vectors has generally represented the most clinicallysuccessful approach to gene therapy. While viral vectors are currentlythe most efficient gene transfer agents, concerns surrounding theoverall safety of viral vectors, which include the potential forunsolicited immune responses, have resulted in parallel efforts todevelop non-viral alternatives (for leading references, see: Luo et al.Nat. Biotechnol. 18:33-37, 2000; Behr Acc. Chem. Res. 26:274-278, 1993;each of which is incorporated herein by reference). Current alternativesto viral vectors include polymeric delivery systems (Zauner et al. Adv.Drug Del. Rev. 30:97-113, 1998; Kabanov et al. Bioconjugate Chem.6:7-20, 1995; each of which is incorporated herein by reference),liposomal formulations (Miller Angew. Chem. Int. Ed. 37:1768-1785, 1998;Hope et al. Molecular Membrane Technology 15:1-14, 1998; Deshmukh et al.New J. Chem. 21:113-124, 1997; each of which is incorporated herein byreference), and “naked” DNA injection protocols (Sanford TrendsBiotechnol. 6:288-302, 1988; incorporated herein by reference). Whilethese strategies have yet to achieve the clinical effectiveness of viralvectors, the potential safety, processing, and economic benefits offeredby these methods (Anderson Nature 392(Suppl.):25-30, 1996; incorporatedherein by reference) have ignited interest in the continued developmentof non-viral approaches to gene therapy (Boussif et al. Proc. Natl.Acad. Sci. USA 92:7297-7301, 1995; Putnam et al. Macromolecules32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999;Gonzalez et al. Bioconjugate Chem. 10:1068-1074, 1999; Kukowska-Latalloet al. Proc. Natl. Acad. Sci. USA 93:4897-4902, 1996; Tang et al.Bioconjugate Chem. 7:703-714, 1996; Haensler et al. Bioconjugate Chem.4:372-379, 1993; each of which is incorporated herein by reference).

Cationic polymers have been widely used as transfection vectors due tothe facility with which they condense and protect negatively chargedstrands of DNA. Amine-containing polymers such as poly(lysine) (Zauneret al. Adv. Drug Del. Rev. 30:97-113, 1998; Kabanov et al. BioconjugateChem. 6:7-20, 1995; each of which is incorporated herein by reference),poly(ethylene imine) (PEI) (Boussif et al. Proc. Natl. Acad. Sci. USA92:7297-7301, 1995; incorporated herein by reference), andpoly(amidoamine) dendrimers (Kukowska-Latallo et al. Proc. Natl. Acad.Sci. USA 93:4897-4902, 1996; Tang et al. Bioconjugate Chem. 7:703-714,1996; Haensler et al. Bioconjugate Chem. 4:372-379, 1993; each of whichis incorporated herein by reference) are positively-charged atphysiological pH, form ion pairs with nucleic acids, and mediatetransfection in a variety of cell lines. Despite their common use,however, cationic polymers such as poly(lysine) and PEI can besignificantly cytotoxic (Zauner et al. Adv. Drug Del. Rev. 30:97-113,1998; Deshmukh et al. New J. Chem. 21:113-124, 1997; Choksakulnimitr etal. Controlled Release 34:233-241, 1995; Brazeau et al. Pharm. Res.15:680-684, 1998; each of which is incorporated herein by reference). Asa result, the choice of cationic polymer for a gene transfer applicationgenerally requires a trade-off between transfection efficiency andshort- and long-term cytotoxicity. Additionally, the long-termbiocompatibility of these polymers remains an important issue for use intherapeutic applications in vivo, since several of these polymers arenot readily biodegradable (Uhrich Trends Polym. Sci. 5:388-393, 1997;Roberts et al. J. Biomed. Mater. Res. 30:53-65, 1996; each of which isincorporated herein by reference).

In order to develop safe alternatives to existing polymeric vectors andother functionalized biomaterials, degradable polyesters bearingcationic side chains have been developed (Putnam et al. Macromolecules32:3658-3662, 1999; Barrera et al. J. Am. Chem. Soc. 115:11010-11011,1993; Kwon et al. Macromolecules 22:3250-3255, 1989; Lim et al. J. Am.Chem. Soc. 121:5633-5639, 1999; Zhou et al. Macromolecules 23:3399-3406,1990; each of which is incorporated herein by reference). Examples ofthese polyesters include poly(L-lactide-co-L-lysine) (Barrera et al. J.Am. Chem. Soc. 115:11010-11011, 1993; incorporated herein by reference),poly(serine ester) (Zhou et al. Macromolecules 23:3399-3406, 1990; eachof which is incorporated herein by reference), poly(4-hydroxy-L-prolineester) (Putnam et al. Macromolecules 32:3658-3662, 1999.; Lim et al. J.Am. Chem. Soc. 121:5633-5639, 1999; each of which is incorporated hereinby reference), and more recently, poly[α-(4-aminobutyl)-L-glycolicacid]. Poly(4-hydroxy-L-proline ester) andpoly[α-(4-aminobutyl)-L-glycolic acid] were recently demonstrated tocondense plasmid DNA through electrostatic interactions, and to mediategene transfer (Putnam et al. Macromolecules 32:3658-3662, 1999; Lim etal. J. Am. Chem. Soc. 121:5633-5639, 1999; each of which is incorporatedherein by reference).

Importantly, these new polymers are significantly less toxic thanpoly(lysine) and PEI, and they degrade into non-toxic metabolites. It isclear from these investigations that the rational design ofamine-containing polyesters can be a productive route to the developmentof safe, effective transfection vectors. Unfortunately, however, presentsyntheses of these polymers require either the independent preparationof specialized monomers (Barrera et al. J. Am. Chem. Soc.115:11010-11011, 1993; incorporated herein by reference), or the use ofstoichiometric amounts of expensive coupling reagents (Putnam et al.Macromolecules 32:3658-3662, 1999; incorporated herein by reference).Additionally, the amine functionalities in the monomers must beprotected prior to polymerization (Putnam et al. Macromolecules32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999;Gonzalez et al. Bioconjugate Chem. 10:1068-1074, 1999; Barrera et al. J.Am. Chem. Soc. 115:11010-11011, 1993; Kwon et al. Macromolecules22:3250-3255, 1989; each of which is incorporated herein by reference),necessitating additional post-polymerization deprotection steps beforethe polymers can be used as transfection agents.

There exists a continuing need for non-toxic, biodegradable,biocompatible polymers that can be used to transfect nucleic acids andthat are easily prepared efficiently and economically. Such polymerswould have several uses, including the delivery of nucleic acids in genetherapy as well as in the packaging and/or delivery of diagnostic,therapeutic, and prophylactic agents.

SUMMARY OF THE INVENTION

The present invention provides polymers useful in preparing drugdelivery devices and pharmaceutical compositions thereof. The inventionalso provides methods of preparing the polymers and methods of preparingmicrospheres and other pharmaceutical compositions containing theinventive polymers.

The polymers of the present invention are poly(β-amino esters) and saltsthereof. Preferred polymers are biodegradable and biocompatible.Typically, the polymers have one or more tertiary amines in the backboneof the polymer. Preferred polymer have one or two tertiary amines perrepeating backbone unit. The polymers may also be co-polymers in whichone of the components is a poly(β-amino ester). The polymers of theinvention may readily be prepared by condensing bis(secondary amines) orprimary amines with bis(acrylate esters). A typical polymer of theinvention is represented by the formulas below:

where A and B are linkers which may be any substituted or unsubstituted,branched or unbranched chain of carbon atoms or heteroatoms. The groupsR₁ and R₂ may be any of a wide variety of substituents. In aparticularly preferred embodiment, the groups R₁ and/or R₂ form cyclicstructures with the linker A (please see the Detailed Descriptionsection below). Polymers containing such cyclic moieties have thecharacteristic of being more soluble at lower pH. Specifically preferredpolymers are those that are insoluble in aqueous solutions atphysiologic pH (pH 7.2-7.4) and are soluble in aqueous solutions belowphysiologic pH (pH<7.2). Other preferred polymers are those that aresoluble in aqueous solution at physiologic pH (pH 7.2-7.4) and below.

In another aspect, the present invention provides a method of preparingthe inventive polymers. In a preferred embodiment, commerciallyavailable or readily available monomers, bis(secondary amines), primaryamines, and bis(acrylate esters), are subjected to conditions which leadto the conjugate addition of the amine to the bis(acrylate ester). In aparticularly preferred embodiment, each of the monomers is dissolved inan organic solvent (e.g., THF, diethyl ether, methylene chloride,hexanes, etc.), and the resulting solutions are combined and heated fora time sufficient to lead to polymerization of the monomers. Theresulting polymer may then be purified using techniques known in theart.

In yet another aspect of the invention, the polymers are used to formnanometer-scale complexes with nucleic acids. The polynucleotide/polymercomplexes may be formed by adding a solution of polynucleotide to avortexing solution of the polymer at a desired DNA/polymerconcentration. The weight to weight ratio of polynucleotide to polymermay range from 1:0.1 to 1:50, preferably from 1:1 to 1:20, morepreferably from 1:1 to 1:10. The cationic polymers condense thepolynucleotide into soluble particles typically 50-500 nm in size. Thesepolynucleotide/polymer complexes may be used in the delivery ofpolynucleotides to cells. In a particularly preferred embodiment, thesecomplexes are combined with pharmaceutical excipients to formpharmaceutical compositions suitable for delivery to animals includinghumans.

In another aspect of the invention, the polymers are used to encapsulatetherapeutic, diagnostic, and/or prophylactic agents includingpolynucleotides to form microparticles. Typically these microparticlesare an order of magnitude larger than the polynucleotide/polymercomplexes. The microparticles range from 1 micrometer to 500micrometers. In a particularly preferred embodiment, thesemicroparticles allow for the delivery of labile small molecules,proteins, peptides, and/or polynucleotides to an individual. Themicroparticles may be prepared using any of the techniques known in theart to make microparticles, such as, for example, double emulsion andspray drying. In a particularly preferred embodiment, the microparticlescan be used for pH-triggered delivery of the encapsulated contents dueto the pH-responsive nature of the polymers (i.e., being more soluble atlower pH).

DEFINITIONS

The following are chemical terms used in the specification and claims:

The term alkyl as used herein refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. Examples of alkyl radicals include, but are not limitedto, methyl, ethyl, propyl, isopropyl, n-butyl, tert-butyl, n-pentyl,neopentyl, n-hexyl, n-heptyl, n-octyl, n-decyl, n-undecyl, and dodecyl.

The term alkoxy as used herein refers to an alkyl groups, as previouslydefined, attached to the parent molecular moiety through an oxygen atom.Examples include, but are not limited to, methoxy, ethoxy, propoxy,isopropoxy, n-butoxy, tert-butoxy, neopentoxy, and n-hexoxy.

The term alkenyl denotes a monovalent group derived from a hydrocarbonmoiety having at least one carbon-carbon double bond by the removal of asingle hydrogen atom. Alkenyl groups include, for example, ethenyl,propenyl, butenyl, 1-methyl-2-buten-1-yl, and the like.

The term alkynyl as used herein refers to a monovalent group derivedform a hydrocarbon having at least one carbon-carbon triple bond by theremoval of a single hydrogen atom. Representative alkynyl groups includeethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

The term alkylamino, dialkylamino, and trialkylamino as used hereinrefers to one, two, or three, respectively, alkyl groups, as previouslydefined, attached to the parent molecular moiety through a nitrogenatom. The term alkylamino refers to a group having the structure —NHR′wherein R′ is an alkyl group, as previously defined; and the termdialkylamino refers to a group having the structure —NR′R″, wherein R′and R″ are each independently selected from the group consisting ofalkyl groups. The term trialkylamino refers to a group having thestructure —NR′R″R′″, wherein R′, R″, and R′″ are each independentlyselected from the group consisting of alkyl groups. Additionally, R′,R″, and/or R′″ taken together may optionally be —(CH₂)_(k)— where k isan integer from 2 to 6. Example include, but are not limited to,methylamino, dimethylamino, ethylamino, diethylamino,diethylaminocarbonyl, methylethylamino, iso-propylamino, piperidino,trimethylamino, and propylamino.

The terms alkylthioether and thioalkoxyl refer to an alkyl group, aspreviously defined, attached to the parent molecular moiety through asulfur atom.

The term aryl as used herein refers to carbocyclic ring system having atleast one aromatic ring including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl, and the like. Aryl groups can beunsubstituted or substituted with substituents selected from the groupconsisting of branched and unbranched alkyl, alkenyl, alkynyl,haloalkyl, alkoxy, thioalkoxy, amino, alkylamino, dialkylamino,trialkylamino, acylamino, cyano, hydroxy, halo, mercapto, nitro,carboxyaldehyde, carboxy, alkoxycarbonyl, and carboxamide. In addition,substituted aryl groups include tetrafluorophenyl and pentafluorophenyl.

The term carboxylic acid as used herein refers to a group of formula—CO₂H.

The terms halo and halogen as used herein refer to an atom selected fromfluorine, chlorine, bromine, and iodine.

The term heterocyclic, as used herein, refers to a non-aromaticpartially unsaturated or fully saturated 3- to 10-membered ring system,which includes single rings of 3 to 8 atoms in size and bi- andtri-cyclic ring systems which may include aromatic six-membered aryl oraromatic heterocyclic groups fused to a non-aromatic ring. Theseheterocyclic rings include those having from one to three heteroatomsindependently selected from oxygen, sulfur, and nitrogen, in which thenitrogen and sulfur heteroatoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized.

The term aromatic heterocyclic, as used herein, refers to a cyclicaromatic radical having from five to ten ring atoms of which one ringatom is selected from sulfur, oxygen, and nitrogen; zero, one, or tworing atoms are additional heteroatoms independently selected fromsulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon,the radical being joined to the rest of the molecule via any of the ringatoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and thelike.

Specific heterocyclic and aromatic heterocyclic groups that may beincluded in the compounds of the invention include:3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine,4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine,4-(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine,4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine,4-(1,1-dimethylethoxycarbonyl)piperazine,4-(2-(bis-(2-propenyl)amino)ethyl)piperazine,4-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine,4-(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine,4-(2-ethylphenyl)piperazine, 4-(2-fluorophenyl)piperazine,4-(2-hydroxyethyl)piperazine, 4-(2-methoxyethyl)piperazine,4-(2-methoxyphenyl)piperazine, 4-(2-methylphenyl)piperazine,4-(2-methylthiophenyl)piperazine, 4-(2-nitrophenyl)piperazine,4-(2-nitrophenyl)piperazine, 4-(2-phenylethyl)piperazine,4-(2-pyridyl)piperazine, 4-(2-pyrimidinyl)piperazine,4-(2,3-dimethylphenyl)piperazine, 4-(2,4-difluorophenyl)piperazine,4-(2,4-dimethoxyphenyl)piperazine, 4-(2,4-dimethylphenyl)piperazine,4-(2,5-dimethylphenyl)piperazine, 4-(2,6-dimethylphenyl)piperazine,4-(3-chlorophenyl)piperazine, 4-(3-methylphenyl)piperazine,4-(3-trifluoromethylphenyl)piperazine, 4-(3,4-dichlorophenyl)piperazine,4-3,4-dimethoxyphenyl)piperazine, 4-(3,4-dimethylphenyl)piperazine,4-(3,4-methylenedioxyphenyl)piperazine,4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine,4-(3,5-dimethoxyphenyl)piperazine,4-(4-(phenylmethoxy)phenyl)piperazine,4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,4-(4-chloro-3-trifluoromethylphenyl)piperazine,4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine,4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine,4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine,4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine,4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine,4-ethylpiperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine,4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine,4-phenylpiperazine, 4-piperidinylpiperazine,4-(2-furanyl)carbonyl)piperazine,4-((1,3-dioxolan-5-yl)methyl)piperazine,6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane,2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine,1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline,azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine,thiomorpholine, and triazole.

The term carbamoyl, as used herein, refers to an amide group of theformula —CONH₂.

The term carbonyldioxyl, as used herein, refers to a carbonate group ofthe formula —O—CO—OR.

The term hydrocarbon, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstitued. The hydrocarbon may be unsaturated, saturated, branched,unbranched, cyclic, polycyclic, or heterocyclic. Illustrativehydrocarbons include, for example, methyl, ethyl, n-propyl, iso-propyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl,methoxy, diethylamino, and the like. As would be known to one skilled inthis art, all valencies must be satisfied in making any substitutions.

The terms substituted, whether preceded by the term “optionally” or not,and substituent, as used herein, refer to the ability, as appreciated byone skilled in this art, to change one functional group for anotherfunctional group provided that the valency of all atoms is maintained.When more than one position in any given structure may be substitutedwith more than one substituent selected from a specified group, thesubstituent may be either the same or different at every position. Thesubstituents may also be further substituted (e.g., an aryl groupsubstituent may have another substituent off it, such as another arylgroup, which is further substituted with fluorine at one or morepositions).

The term thiohydroxyl, as used herein, refers to a thiol of the formula—SH.

The term ureido, as used herein, refers to a urea groups of the formula—NH—CO—NH₂.

The following are more general terms used throughout the presentapplication:

“Animal”: The term animal, as used herein, refers to humans as well asnon-human animals, including, for example, mammals, birds, reptiles,amphibians, and fish. Preferably, the non-human animal is a mammal(e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, aprimate, or a pig). An animal may be a transgenic animal.

“Associated with”: When two entities are “associated with” one anotheras described herein, they are linked by a direct or indirect covalent ornon-covalent interaction. Preferably, the association is covalent.Desirable non-covalent interactions include hydrogen bonding, van derWaals interactions, hydrophobic interactions, magnetic interactions,electrostatic interactions, etc.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe compounds that are not toxic to cells. Compounds are“biocompatible” if their addition to cells in vitro results in less thanor equal to 20% cell death, and their administration in vivo does notinduce inflammation or other such adverse effects.

“Biodegradable”: As used herein, “biodegradable” compounds are thosethat, when introduced into cells, are broken down by the cellularmachinery or by hydrolysis into components that the cells can eitherreuse or dispose of without significant toxic effect on the cells (i.e.,fewer than about 20% of the cells are killed when the components areadded to cells in vitro). The components preferably do not induceinflammation or other adverse effects in vivo. In certain preferredembodiments, the chemical reactions relied upon to break down thebiodegradable compounds are uncatalyzed.

“Effective amount”: In general, the “effective amount” of an activeagent or drug delivery device refers to the amount necessary to elicitthe desired biological response. As will be appreciated by those ofordinary skill in this art, the effective amount of an agent or devicemay vary depending on such factors as the desired biological endpoint,the agent to be delivered, the composition of the encapsulating matrix,the target tissue, etc. For example, the effective amount ofmicroparticles containing an antigen to be delivered to immunize anindividual is the amount that results in an immune response sufficientto prevent infection with an organism having the administered antigen.

“Peptide” or “protein”: According to the present invention, a “peptide”or “protein” comprises a string of at least three amino acids linkedtogether by peptide bonds. The terms “protein” and “peptide” may be usedinterchangeably. Peptide may refer to an individual peptide or acollection of peptides. Inventive peptides preferably contain onlynatural amino acids, although non-natural amino acids (i.e., compoundsthat do not occur in nature but that can be incorporated into apolypeptide chain; see, for example,http://www.cco.caltech.edu/˜dadgrp/Unnatstruct.gif, which displaysstructures of non-natural amino acids that have been successfullyincorporated into functional ion channels) and/or amino acid analogs asare known in the art may alternatively be employed. Also, one or more ofthe amino acids in an inventive peptide may be modified, for example, bythe addition of a chemical entity such as a carbohydrate group, aphosphate group, a farnesyl group, an isofarnesyl group, a fatty acidgroup, a linker for conjugation, functionalization, or othermodification, etc. In a preferred embodiment, the modifications of thepeptide lead to a more stable peptide (e.g., greater half-life in vivo).These modifications may include cyclization of the peptide, theincorporation of D-amino acids, etc. None of the modifications shouldsubstantially interfere with the desired biological activity of thepeptide.

“Polynucleotide” or “oligonucleotide”: Polynucleotide or oligonucleotiderefers to a polymer of nucleotides. Typically, a polynucleotidecomprises at least three nucleotides. The polymer may include naturalnucleosides (i.e., adenosine, thymidine, guano sine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine,C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemicallymodified bases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose), or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

“Small molecule”: As used herein, the term “small molecule” refers toorganic compounds, whether naturally-occurring or artificially created(e.g., via chemical synthesis) that have relatively low molecular weightand that are not proteins, polypeptides, or nucleic acids. Typically,small molecules have a molecular weight of less than about 1500 g/mol.Also, small molecules typically have multiple carbon-carbon bonds. Knownnaturally-occurring small molecules include, but are not limited to,penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Knownsynthetic small molecules include, but are not limited to, ampicillin,methicillin, sulfamethoxazole, and sulfonamides.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the time profile for the degradation of polymers 1-3 at 37°C. at pH 5.1 and pH 7.4. Degradation is expressed as percent degradationover time based on GPC data.

FIG. 2 shows cytotoxicity profiles of polymers 1-3 and PEI. Viability ofNIH 3T3 cells is expressed as a function of polymer concentration. Themolecular weights of polymers 1, 2, and 3 were 5800, 11300, and 22500,respectively. The molecular weight of the PEI employed was 25000.

FIG. 3 shows the retardation of pCMV-Luc DNA by polymer 1 in agarose gelelectrophoresis. Each lane corresponds to a different DNA/polymer weightratio. The ratios are as follows: 1) 1:0 (DNA only); 2) 1:0.5; 3) 1:1;4) 1:2; 5) 1:3; 6) 1:4; 7) 1:5; 8) 1:6; 9) 1:7; and 10) 1:8.

FIG. 4 shows the average effective diameters of DNA/polymer complexesformed from pCMV-Luc plasmid and polymer 3 (M_(n)=31,000) as a functionof polymer concentration.

FIG. 5 shows average ζ-potentials of DNA/polymer complexes formed frompCMV-Luc plasmid and polymer 3 (M_(n)=31,000) as a function of polymerconcentration. The numbers for each complex correspond to the complexnumbers in FIG. 4.

FIG. 6 is an SEM image of rhodamine/dextran-loaded microspheresfabricated from polymer 1.

FIG. 7 shows the release profiles of rhodamine/dextran from polymer 1and PLGA microspheres at various pH values. The arrows indicate thepoints at which HEPES buffer (pH 7.4) was exchanged with acetate buffer(pH 5.1).

FIG. 8 shows a) a representative fluorescence microscopy image ofrhodamine/dextran-loaded polymer 1 microspheres suspended in HEPESbuffer (pH 7.4). FIG. 8 b shows a sample of loaded polymer 1microspheres at pH 7.4 after addition of acetate buffer (pH 5.1). Thedirection of diffusion of acid is from the top right to the bottom leftof the image (elapsed time≈5 seconds).

FIG. 9 demonstrates the gel electrophoresis assay used to identifyDNA-complexing polymers. Lane annotations correspond to the 70water-soluble members of the screening library. For each polymer, assayswere performed at DNA/polymer ratios of 1:5 (left well) and 1:20 (rightwell). Lanes marked C* contain DNA alone (no polymer) and were used as acontrol.

FIG. 10 shows transfection data as a function of structure for an assayemploying pCMV-Luc (600 ng/well, DNA/polymer=1:20). Light units arearbitrary and not normalized to total cell protein; experiments wereperformed in triplicate (error bars not shown). Black squares representwater-insoluble polymers, white squares represent water-soluble polymersthat did not complex DNA in FIG. 9. The right column (marked “*”)displays values for the following control experiments: no polymer(green), PEI (red), and Lipofectamine (light blue).

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides improved polymeric encapuslation anddelivery systems based on the use of β-amino ester polymers. The sytemsmay be used in the pharmaceutical/drug delivery arts to deliverypolynucleotides, proteins, small molecules, peptides, antigen, drugs,etc. to a patient, tissue, organ, cell, etc.

The β-amino ester polymers of the present invention provide for severaldifferent uses in the drug delivery art. The polymers with theirtertiary amine-containing backbones may be used to complexpolynucleotides and thereby enhance the delivery of polynucleotide andprevent their degradation. The polymers may also be used in theformation of nanoparticles or microparticles containing encapsulatedagents. Due to the polymers' properties of being biocompatible andbiodegradable, these formed particles are also biodegradable andbiocompatible and may be used to provide controlled, sustained releaseof the encapsulated agent. These particles may also be responsive to pHchanges given the fact that these polymers are typically notsubstantially soluble in aqueous solution at physiologic pH but are moresoluble at lower pH.

Polymers

The polymers of the present invention are poly(β-amino esters)containing tertiary amines in their backbones and salts thereof. Themolecular weights of the inventive polymers may range from 5,000 g/molto over 100,000 g/mol, more preferably from 4,000 g/mol to 50,000 g/mol.In a particularly preferred embodiment, the inventive polymers arerelatively non-cytotoxic. In another particularly preferred embodiment,the inventive polymers are biocompatible and biodegradable. In aparticularly preferred embodiment, the polymers of the present inventionhave pK_(a)s in the range of 5.5 to 7.5, more preferably between 6.0 and7.0. In another particularly preferred embodiment, the polymer may bedesigned to have a desired pK_(a) between 3.0 and 9.0, more preferablybetween 5.0 and 8.0. The inventive polymers are particularly attractivefor drug delivery for several reasons: 1) they contain amino groups forinteracting with DNA and other negatively charged agents, for bufferingthe pH, for causing endosomolysis, etc.; 2) they contain degradablepolyester linkages; 3) they can be synthesized from commerciallyavailable starting materials; and 4) they are pH responsive and futuregenerations could be engineered with a desired pK_(a).

The polymers of the present invention can generally be defined by theformula (I):

The linkers A and B are each a chain of atoms covalently linking theamino groups and ester groups, respectively. These linkers may containcarbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.).Typically, these linkers are 1 to 30 atoms long, more preferably 1-15atoms long. The linkers may be substituted with various substituentsincluding, but not limited to, hydrogen atoms, alkyl, alkenyl, alkynl,amino, alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy,halogen, aryl, heterocyclic, aromatic heterocyclic, cyano, amide,carbamoyl, carboxylic acid, ester, thioether, alkylthioether, thiol, andureido groups. As would be appreciated by one of skill in this art, eachof these groups may in turn be substituted. The groups R₁, R₂, R₃, R₄,R₅, R₆, R₇, and R₈ may be any chemical groups including, but not limitedto, hydrogen atoms, alkyl, alkenyl, alkynl, amino, alkylamino,dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen, aryl,heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl, carboxylicacid, ester, alkylthioether, thiol, and ureido groups. In the inventivepolymers, n is an integer ranging from 5 to 10,000, more preferably from10 to 500.

In a particularly preferred embodiment, the bis(secondary amine) is acyclic structure, and the polymer is generally represented by theformula II:

In this embodiment, R₁ and R₂ are directly linked together as shown informula II. Examples of inventive polymers in this embodiment include,but are not limited to formulas III and IV:

As described above in the preceding paragraph, any chemical group thatsatisfies the valency of each atom may be substituted for any hydrogenatom.

In another particularly preferred embodiment, the groups R₁ and/or R₂are covalently bonded to linker A to form one or two cyclic structures.The polymers of the present embodiment are generally represented by theformula V in which both R₁ and R₂ are bonded to linker A to form twocyclic structures:

The cyclic structures may be 3-, 4-, 5-, 6-, 7-, or 8-membered rings orlarger. The rings may contain heteroatoms and be unsaturated. Examplesof polymers of formula V include formulas VI, VII, and VIII:

As described above, any chemical group that satisfies the valency ofeach atom in the molecule may be substituted for any hydrogen atom.

In another embodiment, the polymers of the present invention cangenerally be defined by the formula (IX):

The linker B is a chain of atoms covalently linking the ester groups.The linker may contain carbon atoms or heteroatoms (e.g., nitrogen,oxygen, sulfur, etc.). Typically, the linker is 1 to 30 atoms long, morepreferably 1-15 atoms long. The linker may be substituted with varioussubstituents including, but not limited to, hydrogen atoms, alkyl,alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino,hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic,cyano, amide, carbamoyl, carboxylic acid, ester, thioether,alkylthioether, thiol, and ureido groups. As would be appreciated by oneof skill in this art, each of these groups may in turn be substituted.Each of R1, R3, R4, R5, R6, R7, and R8 may be independently any chemicalgroup including, but not limited to, hydrogen atom, alkyl, alkenyl,alkynyl, amino, alkylamino, dialkylamino, trialkylamino, hydroxyl,alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic, cyano,amide, carbamoyl, carboxylic acid, ester, alkylthioether, thiol, andureido groups. In the inventive polymer, n is an integer ranging from 5to 10,000, more preferably from 10 to 500.

In another embodiment, the bis(acrylate ester) unit in the inventivepolymer is chosen from the following group of bis(acrylate ester) units(A-G):

In certain embodiments, the polymer comprises the bis(acrylate ester) G.

In another embodiment, the amine in the inventive polymer is chosen fromthe following group of amines (1-20):

In certain embodiments, the polymer comprises the amine 5. In otherembodiments, the polymer comprises amine 14.

Particular examples of the polymers of the present invention include:

In a particularly preferred embodiment, one or both of the linkers A andB are polyethylene polymers. In another particularly preferredembodiment, one or both of the linkers A and B are polyethylene glycolpolymers. Other biocompatible, biodegradable polymers may be used as oneor both of the linkers A and B.

In another particularly preferred embodiment, the polymer of the presentinvention is a co-polymer wherein one of the repeating units is apoly(β-amino ester) of the present invention. Other repeating units tobe used in the co-polymer include, but are not limited to, polyethylene,poly(glycolide-co-lactide) (PLGA), polyglycolic acid, polymethacrylate,etc.

Synthesis of Polymers

The inventive polymers may be prepared by any method known in the art.Preferably the polymers are prepared from commercially availablestarting materials. In another preferred embodiment, the polymers areprepared from easily and/or inexpensively prepared starting materials.

In a particularly preferred embodiment, the inventive polymer isprepared via the conjugate addition of bis(secondary amines) tobis(acrylate ester). This reaction scheme is shown below:

Bis(secondary amine) monomers that are useful in the present inventivemethod include, but are not limited to, N,N′-dimethylethylenediamine,piperazine, 2-methylpiperazine, 1,2-bis(N-ethylamino)ethylene, and4,4′-trimethylenedipiperidine. Diacrylate monomers that are useful inthe present invention include, but are not limited to, 1,4-butanedioldiacrylate, 1,4-butanediol dimethacrylate, 1,2-ethanediol diacrylate,1,6-hexanediol diacrylate, 2,5-hexanediol diacrylate, and1,3-propanediol diacrylate. Each of the monomers is dissolved in anorganic solvent (e.g., THF, CH₂Cl₂, MeOH, EtOH, CHCl₃, hexanes, toluene,benzene, CCl₄, glyme, diethyl ether, etc.). The resulting solutions arecombined, and the reaction mixture is heated to yield the desiredpolymer. In a particularly preferred embodiment, the reaction mixture isheated to approximately 50° C. In another particularly preferredembodiment, the reaction mixture is heated to approximately 75° C. Thepolymerization reaction may also be catalyzed. As would be appreciatedby one of skill in this art, the molecular weight of the synthesizedpolymer may be determined by the reaction conditions (e.g., temperature,starting materials, concentration, solvent, etc.) used in the synthesis.

In another particularly preferred embodiment, the inventive polymers areprepared by the conjugate addition of a primary amine to a bis(acrylateester). The use of primary amines rather than bis(secondary amines)allows for a much wider variety of commercially available startingmaterials. The reaction scheme using primary amines rather thansecondary amines is shown below:

Primary amines useful in this method include, but are not limited to,methylamine, ethylamine, isopropylamine, aniline, substituted anilines,and ethanolamine. The bis(acrylate esters) useful in this methodinclude, but are not limited to, 1,4-butanediol diacrylate,1,4-butanediol dimethacrylate, 1,2-ethanediol diacrylate, 1,6-hexanedioldiacrylate, 2,5-hexanediol diacrylate, and 1,3-propanediol diacrylate.Each of the monomers is dissolved in an organic solvent (e.g., THF,CH₂Cl₂, MeOH, EtOH, CHCl₃, hexanes, CCl₄, glyme, diethyl ether, etc.).Organic solvents are preferred due to the susceptibility of polyestersto hydrolysis. The resulting solutions are combined, and the reactionmixture is heated to yield the desired polymer. In a particularlypreferred embodiment, the reaction mixture is maintained at 20° C. Inanother particularly preferred embodiment, the reaction mixture isheated to approximately 50° C. In yet another particularly preferredembodiment, the reaction mixture is heated to approximately 75° C. Thereaction mixture may also be cooled to approximately 0° C. Thepolymerization reaction may also be catalyzed. In another preferredembodiment, one or more types of amine monomers and/or diacrylatemonomers may be used in the polymerization reaction. For example, acombination of ethanolamine and ethylamine may be used to prepare apolymer more hydrophilic than one prepared using ethylamine alone, andalso more hydrophobic than one prepared using ethanolamine alone.

The synthesized polymer may be purified by any technique known in theart including, but not limited to, precipitation, crystallization,chromatography, etc. In a particularly preferred embodiment, the polymeris purified through repeated precipitations in organic solvent (e.g.,diethyl ether, hexane, etc.). In a particularly preferred embodiment,the polymer is isolated as a hydrochloride salt. As would be appreciatedby one of skill in this art, the molecular weight of the synthesizedpolymer and the extent of cross-linking may be determined by thereaction conditions (e.g., temperature, starting materials,concentration, order of addition, solvent, etc.) used in the synthesis(Odian Principles of Polymerization 3rd Ed., New York: John Wiley &Sons, 1991; Stevens Polymer Chemistry: An Introduction 2nd Ed., NewYork: Oxford University Press, 1990; each of which is incorporatedherein by reference).

In one embodiment a library of different polymers is prepared inparallel. A different amine and/or bis(acrylate ester) is added to eachvial in a set of vials used to prepare the library. The array of vialsis incubated at a temperature and length of time sufficient to allowpolymerization of the monomers to occur. In one embodiment, the vialsare incubated at approximately 45° C. for approximately 5 days. Thepolymers may then be isolated and purified using techniques known in theart. The polymers may then be screened using high-throughput techniquesto identify polymers with a desired characteristic (e.g., solubility inwater, solubility at different pH, ability to bind polynucleotides,ability to bind heparin, ability to bind small molecules, ability toform microparticles, ability to increase tranfection efficiency, etc.).In certain embodiments the polymers may be screened for properties orcharacteristics useful in gene therapy (e.g., ability to bindpolynucleotides, increase in transfection efficiency). In otherembodiments the polymers may be screened for properties orcharacteristics useful in the art of tissue engineering (e.g., abilityto support tissue or cell growth, ability to promote cell attachment).

Polynucleotide Complexes

The ability of cationic compounds to interact with negatively chargedpolynucleotides through electrostatic interactions is well known.Cationic polymers such as poly(lysine) have been prepared and studiedfor their ability to complex polynucleotides. However, polymers studiedto date have incorporated amines at the terminal ends of short,conformationally flexible side chains (e.g., poly(lysine)) or accessibleamines on the surface of spherical or globular polyamines (e.g., PEI andPAMAM dendrimers). The interaction of the polymer with thepolynucleotide is thought to at least partially prevent the degradationof the polynucleotide. By neutralizing the charge on the backbone of thepolynucleotide, the neutral or slightly-positively-charged complex isalso able to more easily pass through the hydrophobic membranes (e.g.,cytoplasmic, lysosomal, endosomal, nuclear) of the cell. In aparticularly preferred embodiment, the complex is slightly positivelycharged. In another particularly preferred embodiment, the complex has apositive ζ-potential, more preferably the ζ-potential is between +1 and+30.

The poly(β-amino esters) of the present invention possess tertiaryamines in the backbone of the polymer. Although these amines are morehindered, they are available to interact with a polynucleotide.Polynucleotides or derivatives thereof are contacted with the inventivepolymers under conditions suitable to form polynucleotide/polymercomplexes. The polymer is preferably at least partially protonated so asto form a complex with the negatively charged polynucleotide. In apreferred embodiment, the polynucleotide/polymer complexes formnanoparticles that are useful in the delivery of polynucleotides tocells. In a particularly preferred embodiment, the diameter of thenanoparticles ranges from 50-500 nm, more preferably the diameter of thenanoparticles ranges from 50-200 nm, and most preferably from 90-150 nm.The nanoparticles may be associated with a targeting agent as describedbelow.

Polynucleotide

The polynucleotide to be complexed or encapsulated by the inventivepolymers may be any nucleic acid including but not limited to RNA andDNA. The polynucleotides may be of any size or sequence, and they may besingle- or double-stranded. In certain preferred embodiments, thepolynucleotide is greater than 100 base pairs long. In certain otherpreferred embodiments, the polynucleotide is greater than 1000 basepairs long and may be greater than 10,000 base pairs long. Thepolynucleotide is preferably purified and substantially pure.Preferably, the polynucleotide is greater than 50% pure, more preferablygreater than 75% pure, and most preferably greater than 95% pure. Thepolynucleotide may be provided by any means known in the art. In certainpreferred embodiments, the polynucleotide has been engineered usingrecombinant techniques (for a more detailed description of thesetechniques, please see Ausubel et al. Current Protocols in MolecularBiology (John Wiley & Sons, Inc., New York, 1999); Molecular Cloning: ALaboratory Manual, 2nd Ed., ed. by Sambrook, Fritsch, and Maniatis (ColdSpring Harbor Laboratory Press: 1989); each of which is incorporatedherein by reference). The polynucleotide may also be obtained fromnatural sources and purified from contaminating components foundnormally in nature. The polynucleotide may also be chemicallysynthesized in a laboratory. In a preferred embodiment, thepolynucleotide is synthesized using standard solid phase chemistry.

The polynucleotide may be modified by chemical or biological means. Incertain preferred embodiments, these modifications lead to increasedstability of the polynucleotide. Modifications include methylation,phosphorylation, end-capping, etc.

Derivatives of polynucleotides may also be used in the presentinvention. These derivatives include modifications in the bases, sugars,and/or phosphate linkages of the polynucleotide. Modified bases include,but are not limited to, those found in the following nucleoside analogs:2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C5-bromouridine, C5-fluorouridine,C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine. Modified sugarsinclude, but are not limited to, 2′-fluororibose, ribose,2′-deoxyribose, 3′-azido-2′,3′-dideoxyribose, 2′,3′-dideoxyribose,arabinose (the 2′-epimer of ribose), acyclic sugars, and hexoses. Thenucleosides may be strung together by linkages other than thephosphodiester linkage found in naturally occurring DNA and RNA.Modified linkages include, but are not limited to, phosphorothioate and5′-N-phosphoramidite linkages. Combinations of the various modificationsmay be used in a single polynucleotide. These modified polynucleotidesmay be provided by any means known in the art; however, as will beappreciated by those of skill in this art, the modified polynucleotidesare preferably prepared using synthetic chemistry in vitro.

The polynucleotides to be delivered may be in any form. For example, thepolynucleotide may be a circular plasmid, a linearized plasmid, acosmid, a viral genome, a modified viral genome, an artificialchromosome, etc.

The polynucleotide may be of any sequence. In certain preferredembodiments, the polynucleotide encodes a protein or peptide. Theencoded proteins may be enzymes, structural proteins, receptors, solublereceptors, ion channels, pharmaceutically active proteins, cytokines,interleukins, antibodies, antibody fragments, antigens, coagulationfactors, albumin, growth factors, hormones, insulin, etc. Thepolynucleotide may also comprise regulatory regions to control theexpression of a gene. These regulatory regions may include, but are notlimited to, promoters, enhancer elements, repressor elements, TATA box,ribosomal binding sites, stop site for transcription, etc. In otherparticularly preferred embodiments, the polynucleotide is not intendedto encode a protein. For example, the polynucleotide may be used to fixan error in the genome of the cell being transfected.

The polynucleotide may also be provided as an antisense agent or RNAinterference (RNAi) (Fire et al. Nature 391:806-811, 1998; incorporatedherein by reference). Antisense therapy is meant to include, e.g.,administration or in situ provision of single- or double-strandedoligonucleotides or their derivatives which specifically hybridize,e.g., bind, under cellular conditions, with cellular mRNA and/or genomicDNA, or mutants thereof, so as to inhibit expression of the encodedprotein, e.g., by inhibiting transcription and/or translation (Crooke“Molecular mechanisms of action of antisense drugs” Biochim. Biophys.Acta 1489(1):31-44, 1999; Crooke “Evaluating the mechanism of action ofantiproliferative antisense drugs” Antisense Nucleic Acid Drug Dev.10(2):123-126, discussion 127, 2000; Methods in Enzymology volumes313-314, 1999; each of which is incorporated herein by reference). Thebinding may be by conventional base pair complementarity, or, forexample, in the case of binding to DNA duplexes, through specificinteractions in the major groove of the double helix (i.e., triple helixformation) (Chan et al. J. Mol. Med. 75(4):267-282, 1997; incorporatedherein by reference).

In a particularly preferred embodiment, the polynucleotide to bedelivered comprises a sequence encoding an antigenic peptide or protein.Nanoparticles containing these polynucleotides can be delivered to anindividual to induce an immunologic response sufficient to decrease thechance of a subsequent infection and/or lessen the symptoms associatedwith such an infection. The polynucleotide of these vaccines may becombined with interleukins, interferon, cytokines, and adjuvants such ascholera toxin, alum, Freund's adjuvant, etc. A large number of adjuvantcompounds are known; a useful compendium of many such compounds isprepared by the National Institutes of Health and can be found on theWorld Wide Web (http:/www.niaid.nih.gov/daids/vaccine/pdf/compendiumpdf, incorporated herein by reference; see also Allison Dev. Biol.Stand. 92:3-11, 1998; Unkeless et al. Annu. Rev. Immunol. 6:251-281,1998; and Phillips et al. Vaccine 10:151-158, 1992, each of which isincorporated herein by reference).

The antigenic protein or peptides encoded by the polynucleotide may bederived from such bacterial organisms as Streptococccus pneumoniae,Haemophilus influenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; from suchviruses as smallpox, influenza A and B, respiratory syncytial virus,parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1 and 2,cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus,papillomavirus, poliovirus, mumps, rabies, rubella, coxsackieviruses,equine encephalitis, Japanese encephalitis, yellow fever, Rift Valleyfever, hepatitis A, B, C, D, and E virus, and the like; and from suchfungal, protozoan, and parasitic organisms such as Cryptococcusneoformans, Histoplasma capsulatum, Candida albicans, Candidatropicalis, Nocardia asteroides, Rickettsia ricketsii, Rickettsia typhi,Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydial trachomatis,Plasmodium falciparum, Trypanosoma brucei, Entamoeba histolytica,Toxoplasma gondii, Trichomonas vaginalis, Schistosoma mansoni, and thelike.

Microparticles

The poly(β-amino esters) of the present invention may also be used toform drug delivery devices. The inventive polymers may be used toencapsulate agents including polynucleotides, small molecules, proteins,peptides, metals, organometallic compounds, etc. The inventive polymershave several properties that make them particularly suitable in thepreparation of drug delivery devices. These include 1) the ability ofthe polymer to complex and “protect” labile agents; 2) the ability tobuffer the pH in the endosome; 3) the ability to act as a “protonsponge” and cause endosomolysis; and 4) the ability to neutralize thecharge on negatively charged agents. In a preferred embodiment, thepolymers are used to form microparticles containing the agent to bedelivered. In a particularly preferred embodiment, the diameter of themicroparticles ranges from between 500 nm to 50 micrometers, morepreferably from 1 micrometer to 20 micrometers, and most preferably from1 micrometer to 10 micrometers. In another particularly preferredembodiment, the microparticles range from 1-5 micrometers. Theencapsulating inventive polymer may be combined with other polymers(e.g., PEG, PLGA) to form the microspheres.

Methods of Preparing Microparticles

The inventive microparticles may be prepared using any method known inthis art. These include, but are not limited to, spray drying, singleand double emulsion solvent evaporation, solvent extraction, phaseseparation, simple and complex coacervation, and other methods wellknown to those of ordinary skill in the art. Particularly preferredmethods of preparing the particles are the double emulsion process andspray drying. The conditions used in preparing the microparticles may bealtered to yield particles of a desired size or property (e.g.,hydrophobicity, hydrophilicity, external morphology, “stickiness”,shape, etc.). The method of preparing the particle and the conditions(e.g., solvent, temperature, concentration, air flow rate, etc.) usedmay also depend on the agent being encapsulated and/or the compositionof the polymer matrix.

Methods developed for making microparticles for delivery of encapsulatedagents are described in the literature (for example, please see Doubrow,M., Ed., “Microcapsules and Nanoparticles in Medicine and Pharmacy,” CRCPress, Boca Raton, 1992; Mathiowitz and Langer, J. Controlled Release5:13-22, 1987; Mathiowitz et al. Reactive Polymers 6:275-283, 1987;Mathiowitz et al. J. Appl. Polymer Sci. 35:755-774, 1988; each of whichis incorporated herein by reference).

If the particles prepared by any of the above methods have a size rangeoutside of the desired range, the particles can be sized, for example,using a sieve.

Agent

The agents to be delivered by the system of the present invention may betherapeutic, diagnostic, or prophylactic agents. Any chemical compoundto be administered to an individual may be delivered using the inventivemicroparticles. The agent may be a small molecule, organometalliccompound, nucleic acid, protein, peptide, polynucleotide, metal, anisotopically labeled chemical compound, drug, vaccine, immunologicalagent, etc.

In a preferred embodiment, the agents are organic compounds withpharmaceutical activity. In another embodiment of the invention, theagent is a clinically used drug. In a particularly preferred embodiment,the drug is an antibiotic, anti-viral agent, anesthetic, steroidalagent, anti-inflammatory agent, anti-neoplastic agent, antigen, vaccine,antibody, decongestant, antihypertensive, sedative, birth control agent,progestational agent, anti-cholinergic, analgesic, anti-depressant,anti-psychotic, β-adrenergic blocking agent, diuretic, cardiovascularactive agent, vasoactive agent, non-steroidal anti-inflammatory agent,nutritional agent, etc.

In a preferred embodiment of the present invention, the agent to bedelivered may be a mixture of agents. For example, a local anestheticmay be delivered in combination with an anti-inflammatory agent such asa steroid. Local anesthetics may also be administered with vasoactiveagents such as epinephrine. To give another example, an antibiotic maybe combined with an inhibitor of the enzyme commonly produced bybacteria to inactivate the antibiotic (e.g., penicillin and clavulanicacid).

Diagnostic agents include gases; metals; commercially available imagingagents used in positron emissions tomography (PET), computer assistedtomography (CAT), single photon emission computerized tomography, x-ray,fluoroscopy, and magnetic resonance imaging (MRI); and contrast agents.Examples of suitable materials for use as contrast agents in MRI includegadolinium chelates, as well as iron, magnesium, manganese, copper, andchromium Examples of materials useful for CAT and x-ray imaging includeiodine-based materials.

Prophylactic agents include, but are not limited to, antibiotics,nutritional supplements, and vaccines. Vaccines may comprise isolatedproteins or peptides, inactivated organisms and viruses, dead organismsand viruses, genetically altered organisms or viruses, and cellextracts. Prophylactic agents may be combined with interleukins,interferon, cytokines, and adjuvants such as cholera toxin, alum,Freund's adjuvant, etc. Prophylactic agents include antigens of suchbacterial organisms as Streptococccus pneumoniae, Haemophilusinfluenzae, Staphylococcus aureus, Streptococcus pyrogenes,Corynebacterium diphtheriae, Listeria monocytogenes, Bacillus anthracis,Clostridium tetani, Clostridium botulinum, Clostridium perfringens,Neisseria meningitidis, Neisseria gonorrhoeae, Streptococcus mutans,Pseudomonas aeruginosa, Salmonella typhi, Haemophilus parainfluenzae,Bordetella pertussis, Francisella tularensis, Yersinia pestis, Vibriocholerae, Legionella pneumophila, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Leptospirosis interrogans,Borrelia burgdorferi, Camphylobacter jejuni, and the like; antigens ofsuch viruses as smallpox, influenza A and B, respiratory syncytialvirus, parainfluenza, measles, HIV, varicella-zoster, herpes simplex 1and 2, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus,adenovirus, papillomavirus, poliovirus, mumps, rabies, rubella,coxsackieviruses, equine encephalitis, Japanese encephalitis, yellowfever, Rift Valley fever, hepatitis A, B, C, D, and E virus, and thelike; antigens of fungal, protozoan, and parasitic organisms such asCryptococcus neoformans, Histoplasma capsulatum, Candida albicans,Candida tropicalis, Nocardia asteroides, Rickettsia ricketsii,Rickettsia typhi, Mycoplasma pneumoniae, Chlamydial psittaci, Chlamydialtrachomatis, Plasmodium falciparum, Trypanosoma brucei, Entamoebahistolytica, Toxoplasma gondii, Trichomonas vaginalis, Schistosomamansoni, and the like. These antigens may be in the form of whole killedorganisms, peptides, proteins, glycoproteins, carbohydrates, orcombinations thereof.

Targeting Agents

The inventive micro- and nanoparticles may be modified to includetargeting agents since it is often desirable to target a particularcell, collection of cells, or tissue. A variety of targeting agents thatdirect pharmaceutical compositions to particular cells are known in theart (see, for example, Cotten et al. Methods Enzym. 217:618, 1993;incorporated herein by reference). The targeting agents may be includedthroughout the particle or may be only on the surface. The targetingagent may be a protein, peptide, carbohydrate, glycoprotein, lipid,small molecule, etc. The targeting agent may be used to target specificcells or tissues or may be used to promote endocytosis or phagocytosisof the particle. Examples of targeting agents include, but are notlimited to, antibodies, fragments of antibodies, low-densitylipoproteins (LDLs), transferrin, asialycoproteins, gp120 envelopeprotein of the human immunodeficiency virus (HIV), carbohydrates,receptor ligands, sialic acid, etc. If the targeting agent is includedthroughout the particle, the targeting agent may be included in themixture that is used to form the particles. If the targeting agent isonly on the surface, the targeting agent may be associated with (i.e.,by covalent, hydrophobic, hydrogen boding, van der Waals, or otherinteractions) the formed particles using standard chemical techniques.

Pharmaceutical Compositions

Once the microparticles or nanoparticles (polymer complexed withpolynucleotide) have been prepared, they may be combined with one ormore pharmaceutical excipients to form a pharmaceutical composition thatis suitable to administer to animals including humans. As would beappreciated by one of skill in this art, the excipients may be chosenbased on the route of administration as described below, the agent beingdelivered, time course of delivery of the agent, etc.

Pharmaceutical compositions of the present invention and for use inaccordance with the present invention may include a pharmaceuticallyacceptable excipient or carrier. As used herein, the term“pharmaceutically acceptable carrier” means a non-toxic, inert solid,semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. Some examples of materials which canserve as pharmaceutically acceptable carriers are sugars such aslactose, glucose, and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols such as propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; detergentssuch as Tween 80; buffering agents such as magnesium hydroxide andaluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline;Ringer's solution; ethyl alcohol; and phosphate buffer solutions, aswell as other non-toxic compatible lubricants such as sodium laurylsulfate and magnesium stearate, as well as coloring agents, releasingagents, coating agents, sweetening, flavoring and perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator. The pharmaceuticalcompositions of this invention can be administered to humans and/or toanimals, orally, rectally, parenterally, intracisternally,intravaginally, intranasally, intraperitoneally, topically (as bypowders, creams, ointments, or drops), bucally, or as an oral or nasalspray.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredients (i.e.,microparticles, nanoparticles, polynucleotide/polymer complexes), theliquid dosage forms may contain inert diluents commonly used in the artsuch as, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor, and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants such as wetting agents,emulsifying and suspending agents, sweetening, flavoring, and perfumingagents.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Ina particularly preferred embodiment, the particles are suspended in acarrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and0.1% (v/v) Tween 80.

The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the particles withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the microparticles.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, the particlesare mixed with at least one inert, pharmaceutically acceptable excipientor carrier such as sodium citrate or dicalcium phosphate and/or a)fillers or extenders such as starches, lactose, sucrose, glucose,mannitol, and silicic acid, b) binders such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone,sucrose, and acacia, c) humectants such as glycerol, d) disintegratingagents such as agar-agar, calcium carbonate, potato or tapioca starch,alginic acid, certain silicates, and sodium carbonate, e) solutionretarding agents such as paraffin, f) absorption accelerators such asquaternary ammonium compounds, g) wetting agents such as, for example,cetyl alcohol and glycerol monostearate, h) absorbents such as kaolinand bentonite clay, and i) lubricants such as talc, calcium stearate,magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate,and mixtures thereof. In the case of capsules, tablets, and pills, thedosage form may also comprise buffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

The solid dosage forms of tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart. They may optionally contain opacifying agents and can also be of acomposition that they release the active ingredient(s) only, orpreferentially, in a certain part of the intestinal tract, optionally,in a delayed manner Examples of embedding compositions which can be usedinclude polymeric substances and waxes.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like.

Dosage forms for topical or transdermal administration of an inventivepharmaceutical composition include ointments, pastes, creams, lotions,gels, powders, solutions, sprays, inhalants, or patches. The particlesare admixed under sterile conditions with a pharmaceutically acceptablecarrier and any needed preservatives or buffers as may be required.Ophthalmic formulation, ear drops, and eye drops are also contemplatedas being within the scope of this invention.

The ointments, pastes, creams, and gels may contain, in addition to theparticles of this invention, excipients such as animal and vegetablefats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives,polyethylene glycols, silicones, bentonites, silicic acid, talc, andzinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to the particles of thisinvention, excipients such as lactose, talc, silicic acid, aluminumhydroxide, calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants suchas chlorofluorohydrocarbons.

Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the microparticles or nanoparticles in a propermedium. Absorption enhancers can also be used to increase the flux ofthe compound across the skin. The rate can be controlled by eitherproviding a rate controlling membrane or by dispersing the particles ina polymer matrix or gel.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 Degradable Poly(β-Amino Esters): Synthesis,Characterization, and Self-Assembly with Plasmid DNA ExperimentalSection

General Considerations. All manipulations involving live cells orsterile materials were performed in a laminar flow using standardsterile technique. ¹H NMR (300.100 MHz) and ¹³C NMR (75.467 MHz) spectrawere recorded on a Varian Mercury spectrometer. All chemical shiftvalues are given in ppm and are referenced with respect to residualproton or carbon signal from solvent. Organic phase gel permeationchromatography (GPC) was performed using a Hewlett Packard 1100 Seriesisocratic pump, a Rheodyne Model 7125 injector with a 100-μL injectionloop, and two PL-Gel mixed-D columns in series (5 μm, 300×7.5 mm,Polymer Laboratories, Amherst, Mass.). THF/0.1 M piperidine was used asthe eluent at a flow rate of 1.0 mL/min Data was collected using anOptilab DSP interferometric refractometer (Wyatt Technology, SantaBarbara, Calif.) and processed using the TriSEC GPC software package(Viscotek Corporation, Houston, Tex.). The molecular weights andpolydispersities of the polymers are reported relative to monodispersedpolystyrene standards. Aqueous phase GPC was performed by AmericanPolymer Standards (Mentor, Ohio) using Ultrahydrogel L and 120A columnsin series (Waters Corporation, Milford, Mass.). Water (1% acetic acid,0.3 M NaCl) was used as the eluent at a flow rate of 1.0 mL/min Data wascollected using a Knauer differential refractometer and processed usingan IBM/PC GPC-PRO3.13 software package (Viscotek Corporation, Houston,Tex.). The molecular weights and polydispersities of the polymers arereported relative to poly(2-vinylpyridine) standards. For cytotoxicityassays, absorbance was measured using a Dynatech Laboratories MR5000microplate reader at 560 nm.Materials. N,N′-dimethylethylenediamine, piperazine, and4,4′-trimethylenedipiperidine were purchased from Aldrich ChemicalCompany (Milwaukee, Wis.). 1,4-butanediol diacrylate was purchased fromAlfa Aesar Organics (Ward Hill, Mass.).(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) waspurchased from Sigma Chemical Company (St. Louis, Mo.). Plasmid DNA(pCMV-Luc) was produced in E. coli (DHSa, a kind gift from Zycos, Inc.,Cambridge, Mass.), isolated with a Qiagen kit, and purified by ethanolprecipitation. NIH 3T3 cells were purchased from American Type CultureCollection (Manassas, Va.) and grown at 37° C., 5% CO₂ in Dulbecco'smodified Eagle's medium, 90%; fetal bovine serum, 10%; penicillin, 100units/mL; streptomycin, 100 μg/mL. All other materials and solvents wereused as received without further purification.General Polymerization Procedure. In a typical experiment,1,4-butanediol diacrylate (0.750 g, 0.714 mL, 3.78 mmol) and diamine(3.78 mmol) were weighed into two separate vials and dissolved in THF (5mL). The solution containing the diamine was added to the diacrylatesolution via pipette. A Teflon-coated stirbar was added, the vial wassealed with a Teflon-lined screw-cap, and the reaction was heated at 50°C. After 48 hours, the reaction was cooled to room temperature anddripped slowly into vigorously stiffing diethyl ether or hexanes.Polymer was collected and dried under vacuum prior to analysis.Synthesis of Polymer 1. Polymer 1 was prepared according to the generalprocedure outlined above. ¹H NMR δ (CDCl₃, 300 MHz) 4.11 (br t, 4H),2.75 (br t, J=7.05 Hz, 4H), 2.53 (br s, 4H), 2.50 (br t, (obsc), J=7.20Hz, 4H), 2.28 (br s, 6H), 1.71, (br m, 4H). ¹³C NMR δ (CDCl₃, 75.47 MHz)172.55, 64.14, 55.31, 53.39, 42.47, 32.54, 25.53.Synthesis of Polymer 2. Polymer 2 was prepared according to the generalprocedure outlined above. ¹H NMR δ (CDCl₃, 300 MHz) 4.11 (br t, 4H),2.74 (br t, J=7.35, 4H), 2.56 (br m, 12H), 1.71 (br t, 4H). ¹³C NMR δ(CDCl₃, 75.47 MHz) 172.24, 64.19, 53.55, 52.75, 32.27, 25.52.Synthesis of Polymer 3. Polymer 3 was prepared according to the generalprocedure outlined above. ¹H NMR δ (CDCl₃, 300 MHz) 4.11 (br t, 4H),3.00 (br m, 4H), 2.79 (br m, 4H), 2.65 (br m, 4H), 2.11 (br m, 4H), 1.70(br m, 8H), 1.25 (br m, 12H). ¹³C NMR δ (CDCl₃, 75.47 MHz) 172.37,64.13, 53.89 (br), 36.74, 35.58, 32.11 (br), 25.45, 24.05.Polymer Degradation Studies. The hydrochloride salts of polymers 1-3were dissolved in acetate buffer (1 M, pH=5.1) or HEPES buffer (1 M,pH=7.4) at a concentration of 5 mg/mL (the use of millimolarconcentrations of buffer resulted in substantial reduction of pH duringdegradation due to the production of acidic degradation products).Samples were incubated at 37° C. on a mechanical rotator, and aliquots(1 mL) were removed at appropriate time intervals. Aliquots were frozenimmediately in liquid nitrogen and lyophilized. Polymer was extractedfrom dried buffer salts using THF/0.1 M piperidine (1 mL), and sampleswere analyzed directly by GPC.

Formation of DNA/Polymer Complexes and Agarose Gel Retardation Assays.

DNA/polymer complexes were formed by adding 50 μL of a plasmid DNAsolution (pCMV Luc, 2 μg/50 μdL in water) to a gently vortexing solutionof the hydrochloride salt of polymers 1-3 (50 μL in 25 mM MES, pH=6.0,concentrations adjusted to yield desired DNA/polymer weight ratios). Thesamples were incubated at room temperature for 30 minutes, after which20 μL was run on a 1% agarose gel (HEPES, 20 mM, pH=7.2, 65V, 30 mM).Samples were loaded on the gel with a loading buffer consisting of 10%Ficoll 400 (Amersham Pharmacia Biotech, Uppsala, Sweden) in HEPES (25mM, pH=7.2). Bromphenol blue was not included as a visual indicator inthe loading buffer, since this charged dye appeared to interfere withthe complexation of polymer and DNA. DNA bands were visualized under UVillumination by ethidium bromide staining.Quasi-Elastic Laser Light Scattering (QELS) and Measurement ofζ-potentials. QELS experiments and ζ-potential measurements were madeusing a ZetaPALS dynamic light scattering detector (BrookhavenInstruments Corporation, Holtsville, N.Y., 15 mW laser, incidentbeam=676 nm). DNA/polymer complexes were formed as described above foragarose gel retardation assays. Samples were diluted with 900 μL ofHEPES (20 mM, pH=7.0), added to a gently vortexing sample of DNA/polymercomplex (total volume=1 mL, pH=7.0). Average particle sizes andζ-potentials were determined at 25° C. Correlation functions werecollected at a scattering angle of 90°, and particle sizes werecalculated using the MAS option of BIC's particle sizing software (ver.2.30) using the viscosity and refractive index of pure water at 25° C.Particle sizes are expressed as effective diameters assuming a lognormaldistribution. Average electrophoretic mobilities were measured at 25° C.using BIC PALS zeta potential analysis software and zeta potentials werecalculated using the Smoluchowsky model for aqueous suspensions. Threemeasurements were made on each sample, and the results are reported asaverage diameters and zeta potentials.Cytotoxicity Assays. Immortalized NIH 3T3 cells were grown in 96-wellplates at an initial seeding density of 10,000 cells/well in 200 μLgrowth medium (90% Dulbecco's modified Eagle's medium, 10% fetal bovineserum, penicillin 100 units/mL, streptomycin 100 μg/mL) Cells were grownfor 24 hours, after which the growth medium was removed and replacedwith 180 μL of serum-free medium Appropriate amounts of polymer wereadded in 20 μL aliquots. Samples were incubated at 37° C. for 5 hours,and the metabolic activity of each well was determined using aMTT/thiazolyl blue assay: to each well was added 25 μL of a 5 mg/mLsolution of MTT stock solution in sterile PBS buffer. The samples wereincubated at 37° C. for 2 hours, and 100 μL of extraction buffer (20%w/v SDS in DMF/water (1:1), pH=4.7) was added to each well. Samples wereincubated at 37° C. for 24 hours. Optical absorbance was measured at 560nm with a microplate reader and expressed as a percent relative tocontrol cells.

Results and Discussion Polymer Synthesis and Characterization

The synthesis of linear poly(amido amines) containing tertiary amines intheir backbones was reported by Ferruti et al. in 1970 via the additionof bifunctional amines to bisacrylamides (Anderson Nature392(Suppl.):25-30, 1996; Friedman Nature Med. 2:144-147, 1996; CrystalScience 270:404-410, 1995; Mulligan Science 260:926-932, 1993; each ofwhich is incorporated herein by reference). Linear poly(amido amines)were initially investigated as heparin and ion complexing biomaterials(Ferruti et al. Advances in Polymer Science 58:55-92, 1984; Ferruti etal. Biomaterials 15:1235-1241, 1994; Ferruti et al. Macromol. Chem.Phys. 200:1644-1654, 1999; Ferruti et al. Biomaterials 15:1235-1241,1994; each of which is incorporated herein by reference). Dendriticpoly(amido amines) (PAMAMs) have seen increasing use in gene transferapplications due to their ability to complex DNA (Kukowska-Latallo etal. Proc. Natl. Acad. Sci. USA 93:4897-4902, 1996; Tang et al.Bioconjugate Chem. 7:703-714, 1996; Haensler et al. Bioconjugate Chem.4:372-379, 1993; each of which is incorporated herein by reference), anda recent report describes the application of a family of linearpoly(amido amines) to cell transfection and cytotoxicity studies (Hillet al. Biochim. Biophys. Acta 1427:161-174, 1999; incorporated herein byreference). In contrast, analogous poly(ester amines) formed from theMichael-type addition of bifunctional amines to diacrylate esters havereceived less attention (Danusso et al. Polymer 11:88-113, 1970; Ferrutiet al. Polymer 26:1336, 1985; Ferruti et al. Advances in Polymer Science58:55-92, 1984; Ferruti et al. Biomaterials 15:1235-1241, 1994; Ferrutiet al. Macromol. Chem. Phys. 200:1644-1654, 1999; Ferruti et al.Biomaterials 15:1235-1241, 1994; Kargina et al. Vysokomol. Soedin.Seriya A 28:1139-1144, 1986; Rao et al. J. Bioactive and CompatiblePolymers 14:54-63, 1999; each of which is incorporated herein byreference).

The poly(amino ester) approach presents a particularly attractive basisfor the development of new polymeric transfection vectors for severalreasons: 1) the polymers contain the requisite amines and readilydegradable linkages, 2) multiple analogs could potentially besynthesized directly from commercially available starting materials, and3) if the resulting polymers were useful as DNA condensing agents,future generations of polymer could easily be engineered to possessamine pK_(a) values spanning the range of physiologically relevant pH.This last point was particularly intriguing, because the bufferingcapacity of polyamines has recently been implicated as a factorinfluencing the escape of DNA from cell endosomes following endocytosis(Boussif et al. Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995; Haensleret al. Bioconjugate Chem. 4:372-379, 1993; Behr Chimia 51:34-36, 1997;Demeneix et al., in Artificial Self-Assembling Systems for Gene Delivery(Felgner et al., Eds.), American Chemical Society, Washington, D.C.,1996, pp. 146-151; Kabanov et al., in Self-Assembling Complexes for GeneDelivery From Laboratory to Clinical Trial, John Wiley and Sons, NewYork, 1998; each of which is incorporated herein by reference). Whilethe complexation of DNA with a cationic polymer is required to compactand protect DNA during early events in the transfection process, DNA andpolymer must ultimately decomplex in the nucleus to allow efficienttranscription (Luo et al. Nat. Biotechnol. 18:33-37, 2000; incorporatedherein by reference). In view of this requirement, degradablepolycations could play an important role in “vector unpackaging” eventsin the nucleus (Luo et al. Nat. Biotechnol. 18:33-37, 2000; Schaffer etal. Biotechnol. Bioeng. 67:598-606, 2000; Kabanov Pharm. Sci. Technol.Today 2:365-372, 1999; each of which is incorporated herein byreference). Finally, we hypothesized that polymers of this generalstructure, and the β-amino acid derivatives into which they wouldpresumably degrade, would be significantly less toxic than poly(lysine)and PEI. As outlined above, degradable polycations (Putnam et al.Macromolecules 32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc.121:5633-5639, 1999; Lim et al. J. Am. Chem. Soc. 122:6524-6525, 2000;each of which is incorporated herein by reference) and linear polymerscontaining relatively hindered amines located close to the polymerbackbone (Gonzalez et al. Bioconjugate Chem. 10:1068-1074, 1999;incorporated herein by reference) are less toxic than poly(lysine) andPEI.

The synthesis of polymers 1-3 via the addition of the bis(secondaryamines), N,N′-dimethylethylenediamine, piperazine, and4,4′-trimethylenedipiperidine, to 1,4-butanediol diacrylate wasinvestigated (Danusso et al. Polymer 11:88-113, 1970; Kargina et al.Vysokomol. Soedin. Seriya A 28:1139-1144, 1986; each of which isincorporated herein by reference). The polymerization of these monomersproceeded in THF and CH₂Cl₂ at 50° C. to yield the correspondingpolymers in up to 86% yields (Table 1). Polymers were purified throughrepeated precipitation into diethyl ether or hexane. Polymer 1 wasisolated as a clear viscous liquid; polymers 2 and 3 were obtained aswhite solids after drying under high vacuum Alternatively, polymers 1-3could be isolated as solid hydrochloride salts upon addition of diethylether/HCl to a solution of polymer in THF or CH₂Cl₂. All three polymerswere soluble in organic solvents such as THF, CH₂Cl₂, CHCl₃, and MeOHand were also soluble in water at reduced pH. Polymer 1 and thehydrochloride salts of polymers 1-3 were freely soluble in water.

The molecular weights of polymers 1-3 and their correspondinghydrochloride salts were determined by both organic and aqueous phasegel permeation chromatography (GPC). Polymer molecular weights (MOranged from up to 5,800 for polymer 1 to up to 32,000 for polymer 3,relative to polystyrene standards. Molecular weight distributions forthese polymers were monomodal with polydispersity indices (PDIs) rangingfrom 1.55 to 2.55. Representative molecular weight data are presented inTable 1. While the synthesis of linear poly(amido amines) is generallyperformed using alcohols or water as solvents (Danusso et al. Polymer11:88-113, 1970; Ferruti et al. Polymer 26:1336, 1985; Ferruti et al.Advances in Polymer Science 58:55-92, 1984; Ferruti et al. Biomaterials15:1235-1241, 1994; Ferruti et al. Macromol. Chem. Phys. 200:1644-1654,1999; Ferruti et al. Biomaterials 15:1235-1241, 1994; each of which isincorporated herein by reference), anhydrous THF and CH₂Cl₂ wereemployed in the synthesis of poly(β-amino esters) to minimize hydrolysisreactions during the synthesis. The yields and molecular weights ofpolymers synthesized employing CH₂Cl₂ as solvent were generally higherthan those of polymers synthesized in THF (Table 1) (Polymer 1 could notby synthesized in CH₂Cl₂. The color of the reaction solution progressedfrom colorless to an intense pink color almost immediately after theintroduction of a solution of N,N′-dimethylethylenediamine in CH₂Cl₂ toa solution of 1,4-butanediol diacrylate in CH₂Cl₂ (see ExperimentalSection above). The color progressed to light orange over the course ofthe reaction, and an orange polymer was isolated after precipitationinto hexane. The isolated polymer was insoluble in CH₂Cl₂, THF, andwater at reduced pH and was not structurally characterized. This problemwas not encountered for the analogous reaction in THF.).

TABLE 1 Representative Molecular Weight Data for Polymers 1-3. PolymerSolvent M_(n) ^(c) PDI Yield, % 1^(a) THF — — —^(d) 1^(a) CH₂Cl₂ — — 82%2^(a) THF 10 000 1.77 64% 2^(a) CH₂Cl₂ 17 500 2.15 75% 3^(a) THF 24 4001.55 58% 3^(a) CH₂Cl₂ 30 800 2.02 70% 1^(b) THF  5 800 2.83 55% 2^(b)CH₂Cl₂ 16 500 2.37  80%^(e) 3^(b) CH₂Cl₂ 31 200 2.55  86%^(e)^(a)Conditions: [diamine] = [1,4-butanediol diacrylate] = 0.38M, 50° C.,48 h. ^(b)Conditions: [diamine] = [1,4-butanediol diacrylate] = 1.08M,50° C., 48 h. ^(c)GPC analysis was performed in THF/0.1M piperidine andmolecular weights are reported versus polystyrene standards. ^(d)Nopolymer was isolated under these conditions. ^(e)The reaction solutionbecame very viscous and eventually solidified under these conditions.

The structures of polymers 1-3 were confirmed by ¹H and ¹³C NMRspectroscopy. These data indicate that the polymers were formed throughthe conjugate addition of the secondary amines to the acrylate moietiesof 1,4-butanediol diacrylate and not through the formation of amidelinkages under our reaction conditions. Additionally, the newly formedtertiary amines in the polymer backbones do not participate insubsequent addition reactions with diacrylate monomer, which would leadto branching or polymer crosslinking. This fortunate result appears tobe unique to polymers of this type produced from bis(secondary amine)monomers. The synthesis of analogous polymers employing difunctionalprimary amines as monomers (such as 1,4-diaminobutane) may lead topolymer branching and the formation of insoluble crosslinked polymernetworks if conditions are not explicitly controlled.

In view of the juxtaposition of amines and esters within the backbonesof polymers 1-3, we were initially concerned that hydrolysis might occurtoo rapidly for the polymers to be of practical use. For example,poly(4-hydroxy-L-proline ester) and poly[α-(4-aminobutyl)-L-glycolicacid] degrade quite rapidly near neutral pH, having half lives ofroughly 2 hr (Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999;incorporated herein by reference) and 30 min (Lim et al. J. Am. Chem.Soc. 122:6524-6525, 2000; incorporated herein by reference),respectively (Such rapid degradation times did not preclude theapplication of these polymers to gene delivery (See references, Lim etal. J. Am. Chem. Soc. 121:5633-5639, 1999; Lim et al. J. Am. Chem. Soc.122:6524-6525, 2000; each of which is incorporated herein by reference).However, extremely rapid degradation rates generally introduceadditional concerns surrounding the manipulation, storage, andapplication of degradable polymers.). Analysis of polymers 1 and 2 byaqueous GPC using 1% acetic acid/water as eluent, however, revealed thatdegradation was sufficiently slow in acidic media. For example, the GPCtraces of polymers 1 and 2 sampled under these conditions over a periodof 4-5 hours revealed no changes in molecular weights orpolydispersities (Polymer 3 could not be analyzed by aqueous GPC.). Wewere also concerned that significant backbone hydrolysis might occurduring the isolation of the hydrochloride salts of polymers 1-3. Toprevent hydrolysis during the protonation and isolation of thesepolymers, anhydrous solvents were employed and reactions were performedunder an argon atmosphere. Analysis of the polymers before and afterprotonation revealed no observable hydrolysis. For example, the GPCtrace of a sample of polymer 3 after precipitation from CH₂Cl₂ with 1.0M diethyl ether/HCl (M_(n)=15,300; PDI=1.90) was virtually identical tothe molecular weight of the polymer prior to protonation (M_(n)=15,700;PDI=1.92) and no lower molecular weight species were evident(Comparative GPC data were collected employing THF/0.1M piperidine aseluent (see Experimental Section above). The HCl salts of the polymerswere insoluble in THF, but were soluble in THF/0.1 M piperidineconcomitant with the production of a fine white precipitate which wasfiltered prior to injection.). Solid samples of polymers 1-3 could bestored for several months without detectable decreases in molecularweight.

Polymers 1-3 were specifically designed to degrade via hydrolysis of theester bonds in the polymer backbones. However, an additional concernsurrounding the overall stability and biocompatibility of these polymersis the potential for unwanted degradation to occur through retro-Michaelreaction under physiological conditions. Because these polymers weresynthesized via the Michael-type reaction of a secondary amine to anacrylate ester, it is possible that the polymers could undergoretro-Michael reaction to regenerate free acrylate groups, particularlyunder acidic conditions. Acrylate esters are potential DNA-alkylatingagents and are therefore suspected carcinogens (for recent examples,see: Schweikl et al. Mutat. Res. 438:P71-P78, 1999; Yang et al.Carcinogenesis 19:P1117-P1125, 1998; each of which is incorporatedherein by reference). Because these polymers are expected to encounterthe reduced pH environment within the endosomal vesicles of cells(pH=5.0-5.5) during transfection, we addressed the potential for thedegradation of these polymers to occur through a retro-Michael pathway.

Under extremely acidic (pH<3) or basic (pH>12) conditions, polymers 1-3degraded rapidly and exclusively to 1,4-butanediol and the anticipatedbis(β-amino acid) byproducts 4a-6a as determined by ¹H NMR spectroscopy.No spectroscopic evidence for retro-Michael addition under theseconditions was found. It is worth noting that bis(β-amino acid)degradation products 4a-6a would be less likely to undergo aretro-Michael reaction, as acrylic acids are generally less activatedMichael addition partners (Perlmutter, P., in Conjugate AdditionReactions in Organic Synthesis, Pergamon Press, New York, 1992;incorporated herein by reference). Further degradation of compounds4a-6a under these conditions was not observed.

The kinetics of polymer degradation were investigated under the range ofconditions likely to be encountered by these polymers duringtransfection. Degradation was monitored at 37° C. at buffered pH valuesof 5.1 and 7.4 in order to approximate the pH of the environments withinendosomal vesicles and the cytoplasm, respectively. The hydrochloridesalts of polymers 1-3 were added to the appropriate buffer, incubated at37° C., and aliquots were removed at appropriate times. Aliquots werefrozen immediately, lyophilized, and polymer was extracted into THF/0.1M piperidine for analysis by GPC. FIG. 1 shows the degradation profilesof polymers 1-3 as a function of time. The polymers degraded more slowlyat pH 5.1 than at pH 7.4. Polymers 1-3 displayed similar degradationprofiles at pH 5.1, each polymer having a half-life of approximately 7-8hours. In contrast, polymers 1 and 3 were completely degraded in lessthan 5 hours at pH 7.4. These results are consistent with thepH-degradation profiles of other amine-containing polyesters, such aspoly(4-hydroxy-L-proline ester), in which pendant amine functionalitiesare hypothesized to act as intramolecular nucleophilic catalysts andcontribute to more rapid degradation at higher pH (Lim et al. J. Am.Chem. Soc. 121:5633-5639, 1999; Lim et al. J. Am. Chem. Soc.122:6524-6525, 2000; each of which is incorporated herein by reference).

While the possibility of intramolecular assistance cannot be ruled out,it is less likely for polymers 1-3 because the tertiary amines in thesepolymers should be less nucleophilic. The degradation of polymer 2occurred more slowly at pH 7.4 than at pH 5.1 (FIG. 1). This anomalousbehavior is most likely due to the incomplete solubility of polymer 2 atpH 7.4 and the resulting heterogeneous nature of the degradation milieu(Polymers 2 and 3 are not completely soluble in water at pH 7.4. Whilepolymer 3 dissolved relatively rapidly during the degradationexperiment, solid particles of polymer 2 were visible for several days.

Cytotoxicity Assays

Poly(lysine) and PEI have been widely studied as DNA condensing agentsand transfection vectors (Luo et al. Nat. Biotechnol. 18:33-37, 2000;Behr Acc. Chem. Res. 26:274-278, 1993; Zauner et al. Adv. Drug Del. Rev.30:97-113, 1998; Kabanov et al. Bioconjugate Chem. 6:7-20, 1995; Boussifet al. Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995; Behr Chimia51:34-36, 1997; Demeneix et al., in Artificial Self-Assembling Systemsfor Gene Delivery (Felgner et al., Eds.), American Chemical Society,Washington, D.C., 1996, pp. 146-151; Kabanov et al., in Self-AssemblingComplexes for Gene Delivery: From Laboratory to Clinical Trial, JohnWiley and Sons, New York, 1998; each of which is incorporated herein byreference) and are the standards to which new polymeric vectors areoften compared (Putnam et al. Macromolecules 32:3658-3662, 1999; Lim etal. J. Am. Chem. Soc. 121:5633-5639, 1999; Lim et al. J. Am. Chem. Soc.122:6524-6525, 2000; Gonzalez et al. Bioconjugate Chem. 10:1068-1074,1999; each of which is incorporated herein by reference). Unfortunately,as outlined above, these polymers are also associated with significantlevels of cytotoxicity and high levels of gene expression are usuallyrealized only at a substantial cost to cell viability. To determine thetoxicity profile of polymers 1-3, a MTT/thiazolyl blue dye reductionassay using the NIH 3T3 cell line and the hydrochloride salts ofpolymers 1-3 was conducted as an initial indicators. The 3T3 cell lineis commonly employed as a first level screening population for newtransfection vectors, and the MTT assay is generally used as an initialindicator of cytotoxicity, as it determines the influences of addedsubstances on cell growth and metabolism (Hansen et al. Immunol. Methods119:203-210, 1989; incorporated herein by reference).

Cells were incubated with polymer 1 (M_(n)=5 800), polymer 2 (M_(n)=11300), and polymer 3 (M_(n)=22 500) as described in the ExperimentalSection. As shown in FIG. 2, cells incubated with these polymersremained 100% viable relative to controls at concentrations of polymerup to 100 μg/mL These results compare impressively to data obtained forcell populations treated with PEI (M_(n)≈25 000), included as a positivecontrol for our assay as well as to facilitate comparison to thiswell-known transfection agent. Fewer than 30% of cells treated with PEIremained viable at a polymer concentration of 25 μg/mL, and cellviability was as low as 10% at higher concentrations of PEI underotherwise identical conditions. An analogous MTT assay was performedusing independently synthesized bis(β-amino acid)s 4a-6a to screen thecytotoxicity of the hydrolytic degradation products of these polymers.(Bis(β-amino acid)s 4a-6a should either be biologically inert or possessmild or acute toxicities which are lower than traditional polycationictransfection vectors. In either case, the degradation of these materialsshould facilitate rapid metabolic clearance.). Compounds 4a-6a and1,4-butanediol did not perturb cell growth or metabolism in this initialscreening assay (data not shown). A more direct structure/function-basedcomparison between polymers 1-3 and PEI cannot be made due todifferences in polymer structure and molecular weight, both of whichcontribute to polycation toxicity. Nonetheless, the excellentcytotoxicity profiles of polymers 1-3 alone suggested that they wereinteresting candidates for further study as DNA condensing agents.

Self Assembly of Polymers 1-3 with Plasmid DNA

The tendency of cationic polyamines to interact electrostatically withthe polyanionic backbone of DNA in aqueous solution is well known.Provided that the polymers are sufficiently protonated at physiologicalpH, and that the amines are sterically accessible, such interactions canresult in a self-assembly process in which the positively and negativelycharged polymers form well-defined conjugates (Kabanov et al., inSelf-Assembling Complexes for Gene Delivery: From Laboratory to ClinicalTrial, John Wiley and Sons, New York, 1998; each of which isincorporated herein by reference). The majority of polyaminesinvestigated as DNA-complexing agents and transfection vectors haveincorporated amines at the terminal ends of short, conformationallyflexible side chains (e.g., poly(lysine) and methacrylate/methacrylamidepolymers) (Zauner et al. Adv. Drug Del. Rev. 30:97-113, 1998; Kabanov etal. Bioconjugate Chem. 6:7-20, 1995; van de Wetering et al. BioconjugateChem. 10:589-597, 1999; each of which is incorporated herein byreference), or accessible amines on the surfaces of spherical orglobular polyamines (e.g., PEI and PAMAM dendrimers) (Boussif et al.Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995; Kukowska-Latallo et al.Proc. Natl. Acad. Sci. USA 93:4897-4902, 1996; Tang et al. BioconjugateChem. 7:703-714, 1996; Haensler et al. Bioconjugate Chem. 4:372-379,1993; each of which is incorporated herein by reference). Becausepolymers 1-3 contain tertiary amines, and those tertiary amines arelocated in a sterically crowded environment (flanked on two sides by thepolymer backbones), we were initially concerned that the protonatedamines might not be sufficiently able to interact intimately with DNA.

The ability of polymers 1-3 to complex plasmid DNA was demonstratedthrough an agarose gel shift assay. Agarose gel electrophoresisseparates macromolecules on the basis of both charge and size.Therefore, the immobilization of DNA on an agarose gel in the presenceof increasing concentrations of a polycation has been widely used as anassay to determine the point at which complete DNA charge neutralizationis achieved (Putnam et al. Macromolecules 32:3658-3662, 1999; Lim et al.J. Am. Chem. Soc. 121:5633-5639, 1999; Lim et al. J. Am. Chem. Soc.122:6524-6525, 2000; Gonzalez et al. Bioconjugate Chem. 10:1068-1074,1999; each of which is incorporated herein by reference). As mentionedabove, the hydrochloride salts of polymers 1-3 are soluble in water.However, polymers 2 and 3 are not completely soluble at pH 7.2 over thefull range of desired polymer concentrations. Therefore, DNA/polymercomplexes were prepared in MES buffer (25 mM, pH=6.0). DNA/polymercomplexes were prepared by adding an aqueous solution of DNA tovortexing solutions of polymer in MES at desired DNA/polymerconcentrations (see Experimental Section). The resulting DNA/polymercomplexes remained soluble upon dilution in the electrophoresis runningbuffer (20 mM HEPES, pH=7.2) and data were obtained at physiological pH.As a representative example, FIG. 3 depicts the migration of plasmid DNA(pCMV-Luc) on an agarose gel in the presence of increasingconcentrations of polymer 1.

As shown in FIG. 3, retardation of DNA migration begins at DNA/1 ratiosas low as 1:0.5 (w/w) and migration is completely retarded atDNA/polymer ratios above 1:1.0 (w/w) (DNA/polymer weight ratios ratherthan DNA/polymer charge ratios are reported here. Although bothconventions are used in the literature, we find weight ratios to be morepractical and universal, since the overall charge on a polyamine issubject to environmental variations in pH and temperature. WhileDNA/polymer charge ratios are descriptive for polymers such aspoly(lysine), they are less meaningful for polymers such as PEI and 1-3which incorporate less basic amines) Polymers 2 and 3 completely inhibitthe migration of plasmid DNA at DNA/polymer ratios (w/w) above 1:10 and1:1.5, respectively (data not shown). These results vary markedly fromgel retardation experiments conducted using model “monomers.” Since thetrue monomers and the degradation products of polymers 1-3 do notadequately represent the repeat units of the polymers, we usedbis(methyl ester)s 4b-6b to examine the extent to which the polyvalencyand cooperative binding of polycations 1-3 is necessary to achieve DNAimmobilization. “Monomers” 4b-6b did not inhibit the migration of DNA atDNA/“monomer” ratios (w/w) of up to 1:30 (data not shown).

The reasons for the less-efficient complexation employing polymer 2 inthe above gel electrophoresis assays most likely results fromdifferences in the pK_(a) values of the amines in these polymers. Thedirect measurement of the pK_(a) values of polymers 1-3 is complicatedby their degradability. However, we predict the range of pK_(a) valuesof the amines in polymers 1 and 2 to extend from approximately 4.5 and8.0 for polymer 1, to 3.0 and 7.0 for polymer 2, based on comparisons tostructurally related poly(β-amino amides) (The pK_(a) values ofstructurally-related poly(β-amino amides) containing piperazine anddimethylethylene diamine units in their backbones have been reported.Barbucci et al. Polymer 21:81-85, 1980; Barbucci et al. Polymer19:1329-1334, 1978; Barbucci et al. Macromolecules 14:1203-1209, 1981;each of which is incorporated herein by reference). As a result, polymer2 should be protonated to a lesser extent than polymer 1 atphysiological or near-neutral pH, and would therefore be a lesseffective DNA condensing agent. The range of pK_(a) values for polymer 3should be higher than the range for polymers 1 and 2 due to theincreased distance between the nitrogen atoms. Accordingly, polymer 3forms complexes with DNA at substantially reduced concentrationsrelative to polymer 2.

Agarose gel retardation assays are useful in determining the extent towhich polycations interact with DNA. To be useful transfection agents,however, polycations must also be able to self-assemble plasmid DNA intopolymer/DNA complexes small enough to enter a cell through endocytosis.For most cell types, this size requirement is on the order of 200 nm orless (Zauner et al. Adv. Drug Del. Rev. 30:97-113, 1998; incorporatedherein by reference), although larger particles can also be accommodated(Demeneix et al., in Artificial Self-Assembling Systems for GeneDelivery (Felgner et al., Eds.), American Chemical Society, Washington,D.C., 1996, pp. 146-151; Kabanov et al., in Self-Assembling Complexesfor Gene Delivery: From Laboratory to Clinical Trial, John Wiley andSons, New York, 1998; each of which is incorporated herein byreference). The ability of polymers 1-3 to compact plasmid DNA intonanometer-sized structures was determined by quasi-elastic laser lightscattering (QELS), and the relative surface charges of the resultingcomplexes were quantified through ζ-potential measurements. DNA/polymerparticles used for particle sizing and ζ-potential measurements wereformed as described above for agarose gel electrophoresis assays anddiluted in 20 mM HEPES buffer (pH

=7.0) for analysis, as described in the Experimental Section.

Polymer 1 formed complexes with diameters ranging from 90-150 nm atDNA/polymer ratios above 1:2 (w/w), and polymer 2 condensed DNA intoparticles on the order of 60-125 nm at DNA/polymer ratios above 1:10.These results are consistent with the data obtained from agarose gelelectrophoresis experiments above. However, the particles in theseexperiments aggregated over a period of hours to yield larger complexeswith diameters in the range of 1-2 microns. The tendency of theseparticles to aggregate can be rationalized by the low ζ-potentials ofthe DNA/polymer particles observed under these conditions. For example,complexes formed from polymer 1 at DNA/polymer ratios above 1:10 hadaverage ζ-potentials of +4.51 (±0.50) mV. The ζ-potentials of complexesformed from polymer 2 at DNA/polymer ratios above 1:20 were lower,reaching a limiting value of +1.04 (±0.57) mV. These differencescorrelate with the estimated pK_(a) values for these polymers, as thesurfaces of particles formed from polymer 1 would be expected toslightly more protonated than particles formed from polymer 2 at pH=7.0.

Polymer 3 formed complexes with diameters in the range of 50-150 nm atDNA/polymer ratios above 1:2. As a representative example, FIG. 4 showsthe average effective diameters of particles formed with polymer 3 as afunction of polymer concentration. The diameters of the particles variedwithin the above range from experiment to experiment under otherwiseidentical conditions, possibly due to subtle differences during thestirring or addition of DNA solutions during complex formation (Theorder of addition of polymer and DNA solutions had considerable impacton the nature of the resulting DNA/polymer complexes. In order to formsmall particles, for example, it was necessary to add the DNA solutionto a vortexing solution of polymer. For cases in which polymer solutionswere added to DNA, only large micron-sized aggregates were observed.Thus, it is possible that subtle differences in stirring or rate ofaddition could be responsible for variation in particle size). Theζ-potentials for complexes formed from polymer 3 were on the order of+10 to +15 mV at DNA/polymer ratios above 1:1, and the complexes did notaggregate extensively over an 18 hour period (pH=7, 25° C.) The positiveζ-potentials of these complexes may be significant beyond the context ofparticle stability, as net positive charges on particle surfaces mayplay a role in triggering endocytosis (Kabanov et al. Bioconjugate Chem.6:7-20, 1995; Lim et al. J. Am. Chem. Soc. 122:6524-6525, 2000; BehrChimia 51:34-36, 1997; Demeneix et al., in Artificial Self-AssemblingSystems for Gene Delivery (Felgner et al., Eds.), American ChemicalSociety, Washington, D.C., 1996, pp. 146-151; Kabanov et al., inSelf-Assembling Complexes for Gene Delivery: From Laboratory to ClinicalTrial, John Wiley and Sons, New York, 1998; each of which isincorporated herein by reference).

Particles formed from polymer 3 were also relatively stable at 37° C.For example, a sample of DNA/3 (DNA/3=1:5, average diameter=83 nm) wasincubated at 37° C. for 4 hours. After 4 hours, a bimodal distributionwas observed consisting of a fraction averaging 78 nm (>98% by number,70% by volume) and a fraction of larger aggregates with averagediameters of approximately 2.6 microns. These results suggest that thedegradation of complexes formed from polymer 3 occurred more slowly thanthe degradation of polymer in solution, or that partial degradation didnot significantly affect the stability of the particles. The apparentlyincreased stability of DNA/polymer complexes formed from degradablepolycations relative to the degradation of the polymers in solution hasalso been observed for DNA/polymer complexes formed frompoly(4-hydroxy-L-proline ester) (Lim et al. J. Am. Chem. Soc.121:5633-5639, 1999; incorporated herein by reference).

The particle size and ζ-potential data in FIGS. 4 and 5 are consistentwith models of DNA condensation observed with other polycations (Kabanovet al. Bioconjugate Chem. 6:7-20, 1995; Putnam et al. Macromolecules32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999;Lim et al. J. Am. Chem. Soc. 122:6524-6525, 2000; Gonzalez et al.Bioconjugate Chem. 10:1068-1074, 1999; each of which is incorporatedherein by reference). DNA is compacted into small negatively chargedparticles at very low polymer concentrations and particle sizes increasewith increasing polymer concentration (Accurate light scattering datacould not be obtained for DNA alone or for DNA/polymer associatedspecies at DNA/polymer ratios lower than 1:0.5, since flexible,uncondensed DNA does not scatter light as extensively as compacted DNA(Kabanov et al., in Self-Assembling Complexes for Gene Delivery: FromLaboratory to Clinical Trial, John Wiley and Sons, New York, 1998;incorporated herein by reference).). Complexes reach a maximum diameteras charge neutrality is achieved and aggregation occurs. Particle sizesdecrease sharply at DNA/polymer concentrations above charge neutralityup to ratios at which additional polymer does not contribute to areduction in particle diameter. This model is confirmed by ζ-potentialmeasurements made on complexes formed from these polymers. As shown inFIG. 5, the ζ-potentials of polymer/DNA particles formed from polymer 3were negative at low polymer concentrations and charge neutrality wasachieved near DNA/polymer ratios of 1:0.75, resulting in extensiveaggregation. The ζ-potentials of the particles approached a limitingvalue ranging from +10 to +15 mV at DNA/polymer ratios above 1:2.

The average diameters of the complexes described above fall within thegeneral size requirements for cellular endocytosis. We have initiatedtransfection experiments employing the NIH 3T3 cell line and theluciferase reporter gene (pCMV-Luc). Thus far, polymers 1 and 2 haveshown no transfection activity in initial screening assays. By contrast,polymer 3 has demonstrated transfection efficiencies exceeding those ofPEI under certain conditions. Transfection experiments were performedaccording to the following general protocol: Cells were grown in 6-wellplates at an initial seeding density of 100,000 cells/well in 2 mL ofgrowth medium Cells were grown for 24 hours after which the growthmedium was removed and replaced with 2 mL of serum-free mediumDNA/polymer complexes were formed as described in the ExperimentalSection (2 μg DNA, DNA/3=1:2 (w/w), 100 μL in MES (pH=6.0)] and added toeach well. DNA/PEI complexes were formed at a weight ratio of 1:0.75, aratio generally found in our laboratory to be optimal for PEItransfections. Transfections were carried out in serum-free medium for 4hours, after which medium was replaced with growth medium for 20additional hours. Relative transfection efficiencies were determinedusing luciferase (Promega) and cell protein assay (Pierce) kits. Resultsare expressed as relative light units (RLU) per mg of total cellprotein: for complexes of polymer 3, 1.07 (±0.43)×10⁶ RLU/mg; for PEIcomplexes, 8.07 (±0.16)×10⁵ RLU/mg). No luciferase expression wasdetected for control experiments employing naked DNA or performed in theabsence of DNA. These transfection data are the results of initialscreening experiments. These data suggest that polymers of this generalstructure hold promise as synthetic vectors for gene delivery and areinteresting candidates for further study. The relative efficacy ofpolymer 3 relative to PEI is interesting, as our initial screeningexperiments were performed in the absence of chloroquine and polymer 3does not currently incorporate an obvious means of facilitatingendosomal escape. It should be noted, however, that the pK_(a) values ofthe amines in these polymers can be “tuned” to fall more directly withinthe range of physiologically relevant pH using this modular syntheticapproach. Therefore, it will be possible to further engineer the “protonsponge” character (Behr Chimia 51:34-36, 1997; Demeneix et al., inArtificial Self-Assembling Systems for Gene Delivery (Felgner et al.,Eds.), American Chemical Society, Washington, D.C., 1996, pp. 146-151;Kabanov et al., in Self-Assembling Complexes for Gene Delivery: FromLaboratory to Clinical Trial, John Wiley and Sons, New York, 1998; eachof which is incorporated herein by reference) of these polymers, andthus enhance their transfection efficacies, directly through theincorporation of or copolymerization with different diamine monomers.

SUMMARY

A general strategy for the preparation of new degradable polymeric DNAtransfection vectors is reported. Poly(β-amino esters) 1-3 weresynthesized via the conjugate addition of N,N′-dimethylethylenediamine,piperazine, and 4,4′-trimethylenedipiperidine to 1,4-butanedioldiacrylate. The amines in the bis(secondary amine) monomers activelyparticipate in bond-forming processes during polymerization, obviatingthe need for amine protection/deprotection processes which characterizethe synthesis of other poly(amino esters). Accordingly, this approachenables the generation of a variety of structurally diverse polyesterscontaining tertiary amines in their backbones in a single step fromcommercially available staring materials. Polymers 1-3 degradedhydrolytically in acidic and alkaline media to yield 1,4-butanediol andβ-amino acids 4a-6a and the degradation kinetics were investigated at pH5.1 and 7.4. The polymers degraded more rapidly at pH 7.4 than at pH5.1, consistent with the pH/degradation profiles reported for otherpoly(amino esters). An initial screening assay designed to determine theeffects of polymers 1-3 on cell growth and metabolism suggested thatthese polymers and their hydrolytic degradation products werenon-cytotoxic relative to PEI, a non-degradable cationic polymerconventionally employed as a transfection vector.

Polymers 1-3 interacted electrostatically with plasmid DNA atphysiological pH, initiating self-assembly processes that resulted innanometer-scale DNA/polymer complexes. Agarose gel electrophoresis,quasi-elastic dynamic light scattering (QELS), and zeta potentialmeasurements were used to determine the extent of the interactionsbetween the oppositely charged polyelectrolytes. All three polymers werefound to condense DNA into soluble DNA/polymer particles on the order of50-200 nm Particles formed from polymers 1 and 2 aggregated extensively,while particles formed from polymer 3 exhibited positive ζ-potentials(e.g., +10 to +15 mV) and did not aggregate for up to 18 hours. Thenanometer-sized dimensions and reduced cytotoxicities of theseDNA/polymer complexes suggest that polymers 1-3 may be useful asdegradable polymeric gene transfection vectors. A thorough understandingof structure/activity relationships existing for this class of polymerwill expedite the design of safer polymer-based alternatives to viraltransfection vectors for gene therapy.

Example 2 Rapid, pH-Triggered Release from Biodegradable Poly(β-AminoEster) Microspheres within the Ranger of Intracellular pH ExperimentalSection

Fabrication of microspheres. The optimized procedure for the fabricationof microspheres was conducted in the following general manner: Anaqueous solution of rhodamine-conjugated dextran (200 μL of a 10 μg/μLsolution, M_(n)≈70 kD) was suspended in a solution of poly-1 in CH₂Cl₂(200 mg of poly-1 in 4 mL CH₂Cl₂, M_(b≈10) kD), and the mixture wassonicated for 10 seconds to form a primary emulsion. The cloudy pinkemulsion was added directly to a rapidly homogenized (5,000 rpm)solution of poly(vinyl alcohol) [50 mL, 1% PVA (w/w)] to form thesecondary emulsion. The secondary emulsion was homogenized for 30seconds before adding it to a second aqueous PVA solution [100 mL, 0.5%PVA (w/w)]. Direct analysis of the microsphere suspension using aCoulter microparticle analyzer revealed a mean particle size ofapproximately 5 micrometers. The secondary emulsion was stirred for 2.5hours at room temperature, transferred to a cold room (4° C.), andstirred for an additional 30 minutes. Microspheres were isolated at 4°C. via centrifugation, resuspended in cold water, and centrifuged againto remove excess PVA. The spheres were resuspended in water (15 mL) andlyophilized to yield a pink, fluffy powder. Characterization of thelyophilized microspheres was performed by optical, fluorescence, andscanning electron microscopies as described. Zeta potential wasdetermined using a Brookhaven Instruments ZetaPALS analyzer.

Discussion

Microparticles formed from biodegradable polymers are attractive for useas delivery devices, and a variety of polymer-based microspheres havebeen employed for the sustained release of therapeutic compounds(Anderson Nature 392(Suppl.):25-30, 1996; Friedman Nature Med.2:144-147, 1996; Crystal Science 270:404-410, 1995; Mulligan Science260:926-932, 1993; Luo et al. Nat. Biotechnol. 18:33-37, 2000; Behr Acc.Chem. Res. 26:274-278, 1993; each of which is incorporated herein byreference). However, for small-molecule-, protein-, and DNA-basedtherapeutics that require intracellular administration and traffickingto the cytoplasm, there is an increasing demand for new materials thatfacilitate triggered release in response to environmental stimuli suchas pH (Zauner et al. Adv. Drug Del. Rev. 30:97-113, 1998; incorporatedherein by reference). Following endocytosis, the pH within cellularendosomal compartments is lowered, and foreign material is degraded uponfusion with lysosomal vesicles (Kabanov et al. Bioconjugate Chem.6:7-20, 1995; incorporated herein by reference). New materials thatrelease molecular payloads upon changes in pH within the intracellularrange and facilitate escape from hostile intracellular environmentscould have a fundamental and broad-reaching impact on the administrationof hydrolytically- and/or enzymatically-labile drugs (Zauner et al. Adv.Drug Del. Rev. 30:97-113, 1998; Kabanov et al. Bioconjugate Chem.6:7-20, 1995; each of which is incorporated herein by reference).Herein, the fabrication of pH-responsive polymer microspheres thatrelease encapsulated contents quantitatively and essentiallyinstantaneously upon changes in pH within the intracellular range isreported.

The synthesis of poly(β-amino ester) 1 has been described above inExample 1 (Miller Angew. Chem. Int. Ed. 37:1768-1785, 1998; Hope et al.Molecular Membrane Technology 15:1-14, 1998; Deshmukh et al. New J.Chem. 21:113-124, 1997; each of which is incorporated herein byreference). Poly-1 is hydrolytically degradable, was non-cytotoxic ininitial screening assays, and is currently under investigation as asynthetic vector for DNA delivery in gene therapy applications. Thesolubility of the polymer in aqueous media is directly influenced bysolution pH. Specifically, the solid, unprotonated polymer is insolublein aqueous media in the pH range 7.0 to 7.4, and the transition betweensolubility and insolubility occurs at a pH around 6.5. Based on thedifferences between extracellular and endosomal pH (7.4 and 5.0-6.5,respectively), we hypothesized that microspheres formed from poly-1might be useful for the encapsulation and triggered release of compoundswithin the range of intracellular pH.

The encapsulation of therapeutic compounds within polymer microspheresis often achieved employing a double emulsion process (O'Donnell et al.Adv. Drug Delivery Rev. 28:25-42, 1997; incorporated herein byreference). The double emulsion process is well established for thefabrication of microspheres from hydrophobic polymers such aspoly(lactic-co-glycolic acid) (PLGA), a biodegradable polymerconventionally employed in the development of drug delivery devices(Anderson et al. Adv. Drug Delivery Rev. 28:5-24, 1997; Okada Adv. DrugDelivery Rev. 28:43-70, 1997; each of which is incorporated herein byreference). Preliminary experiments demonstrated the feasibility of thedouble emulsion process for the encapsulation of water-soluble compoundsusing poly-1. Rhodamine-conjugated dextran was chosen as a model forsubsequent encapsulation and release studies for several reasons: 1)rhodamine is fluorescent, allowing loading and release profiles to bedetermined by fluorescence spectroscopy, 2) loaded microspheres could beimaged directly by fluorescence microscopy, and 3) the fluorescenceintensity of rhodamine is relatively unaffected by pH within thephysiological range (Haugland, Handbook of Fluorescent Probes andResearch Chemicals, 6th ed., Molecular Probes, Inc., 1996, p. 29;incorporated herein by reference).

Microspheres encapsulating labeled dextran were fabricated from poly-1and compared to controls formed from PLGA. The size distributions ofmicrospheres formed from poly-1 correlated well with the distributionsof PLGA microspheres within the range of 5-30 μm. Average particle sizescould be controlled by variations in experimental parameters such ashomogenization rates and aqueous/organic solvent ratios (O'Donnell etal. Adv. Drug Delivery Rev. 28:25-42, 1997; incorporated herein byreference). In contrast to PLGA microspheres, however, spheres formedfrom poly-1 aggregated extensively during centrifugation and washingsteps (see Experimental Section above). Microspheres resuspended at pH7.4 consisted primarily of large aggregates, and scanning electronmicroscopy (SEM) images revealed clusters of spheres that appeared to bephysically joined or “welded” (data not shown).

It was found that aggregation could be eliminated if centrifugation andwashing were conducted at reduced temperatures (4° C.), presumably dueto the hardening of the polymer spheres at this lower temperature. SEMimages of dextran-loaded poly-1 microspheres prepared in the 8-10 μmrange revealed significant fracturing and the formation of large holeson their surfaces. Microspheres targeted in the range of 4-6 μm,however, were essentially free of cracks, holes, and other defects (FIG.6). Microspheres formulated for subsequent release experiments werefabricated in the smaller (<6 μm) range. Encapsulation efficiencies forloaded poly-1 microspheres, determined by dissolving the spheres at pH5.1 and measuring fluorescence intensity, were as high as 53%.

Suspensions of dried poly-1 microspheres at pH=7.4 consisted primarilyof single, isolated microspheres as determined by optical andfluorescence microscopy (FIG. 8 a). The zeta potential (ζ) ofmicroparticle suspensions of poly-1 microspheres at pH 7 was +3.75(±0.62) mV, suggesting that the surfaces of the microspheres carry anoverall positive charge at physiological pH. This could be relevant tothe targeting of these microspheres for cellular uptake, because netpositive charges on particle surfaces may play a role in triggeringendocytosis (Zauner et al. Adv. Drug Delivery Rev. 30:97-113, 1998;incorporated herein by reference).

Poly-1 microspheres suspended at pH 7.4 remained stable towardaggregation and degradation for several weeks (by visual inspection),but the microspheres dissolved instantly when the pH of the suspendingmedium was lowered between 5.1 and 6.5.

The release of labeled dextran from poly-1 microspheres was determinedquantitatively by fluorescence microscopy (FIG. 7). The release profileat pH 7.4 was characterized by a small initial burst in fluorescence(7-8%) which reached a limiting value of about 15% after 48 hours. Thisexperiment demonstrated that the degradation of poly-1 was relativelyslow under these conditions and that greater than 90% of encapsulatedmaterial could be retained in the polymer matrix for suitably longperiods of time at physiological pH.

To examine the application of poly-1 microspheres to the triggeredrelease of encapsulated drugs in the endosomal pH range, we conducted asimilar experiment in which the pH of the suspension medium was changedfrom 7.4 to 5.1 during the course of the experiment. As shown in FIG. 7,the microspheres dissolved rapidly when the suspension buffer wasexchanged with acetate buffer (0.1 M, pH=5.1), resulting in essentiallyinstantaneous and quantitative release of the labeled dextran remainingin the polymer matrices. In sharp contrast, the release fromdextran-loaded PLGA microspheres did not increase for up to 24 hoursafter the pH of the suspending medium was lowered (FIG. 7). FIG. 8 showsfluorescence microscopy images of: (a) a sample of dextran-loadedmicrospheres at pH 7.4; and (b) a sample to which a drop of acetatebuffer was added at the upper right edge of the microscope coverslip.The rapid release of rhodamine-conjugated dextran was visualized asstreaking extending from the dissolving microspheres in the direction ofthe diffusion of added acid and an overall increase in backgroundfluorescence (elapsed time≈5 seconds).

When targeting therapeutic compounds for intracellular delivery viaendocytosis or phagocytosis, it is not only important to consider ameans by which the drug can be released from its carrier, but also ameans by which the drug can escape endosomal compartments prior to beingrouted to lysosomal vesicles (Luo et al. Nat. Biotechnol. 18:33-37,2000; Zauner et al. Adv. Drug Delivery Rev. 30:97-113, 1998; each ofwhich is incorporated herein by reference). One strategy forfacilitating endosomal escape is the incorporation of weak bases, or“proton sponges,” which are believed to buffer the acidic environmentwithin an endosome and disrupt endosomal membranes by increasing theinternal osmotic pressure within the vesicle (Demeneix et al., inArtificial Self-Assembling Systems for Gene Delivery (Felgner et al.,Eds.), American Chemical Society, Washington, D.C., 1996, pp. 146-151;incorporated herein by reference). Poly-1 microspheres are capable ofreleasing encapsulated material in the endosomal pH range via amechanism (dissolution) that involves the protonation of amines in thepolymer matrix. Thus, in addition to the rapid release of drug, poly-1microspheres may also provide a membrane-disrupting means of endosomalescape, enhancing efficacy by prolonging the lifetimes of hydrolyticallyunstable drugs contained in the polymer matrix.

Microspheres fabricated from poly-1 could represent an importantaddition to the arsenal of pH-responsive materials applied forintracellular drug delivery, such as pH-responsive polymer/liposomeformulations (Gerasimov et al. Adv. Drug Delivery Rev. 38:317-338, 1999;Linhart et al. Langmuir 16:122-127, 2000; Linhardt et al. Macromolecules32:4457-4459, 1999; each of which is incorporated herein by reference).In contrast to many liposomal formulations, polymer microspheres arephysically robust and can be stored dried for extended periods withoutdeformation, decomposition, or degradation (Okada Adv. Drug DeliveryRev. 28:43-70, 1997; incorporated herein by reference)—an importantconsideration for the formulation and packaging of new therapeuticdelivery systems. The microspheres investigated in this current studyfall within the size range of particles commonly used to target deliveryto macrophages (Hanes et al. Adv. Drug Delivery Rev. 28:97-119, 1997;incorporated herein by reference). The rapid pH-release profiles for thepoly-1 microspheres described above may therefore be useful in thedesign of new DNA-based vaccines which currently employ PLGA as anencapsulating material (Singh et al. Proc. Natl. Acad. Sci. USA97:811-816, 2000; Ando et al. J. Pharm. Sci. 88:126-130, 1999; Hedley etal. Nat. Med. 4:365-368, 1998; each of which is incorporated herein byreference).

Example 3 Accelerated Discovery of Synthetic Transfection Vectors:Parallel Synthesis and Screening of a Degradable Polymer LibraryIntroduction

The safe and efficient delivery of therapeutic DNA to cells represents afundamental obstacle to the clinical success of gene therapy (Luo et al.Nat. Biotechnol. 18:33-37, 2000; Anderson Nature 392 Suppl.:25-30, 1996;each of which is incorporated herein by reference). The challengesfacing synthetic delivery vectors are particularly clear, as bothcationic polymers and liposomes are less effective at mediating genetransfer than viral vectors. The incorporation of new design criteriahas led to recent advances toward functional delivery systems (Lim etal. J. Am. Chem. Soc. 123:2460-2461, 2001; Lim et al. J. Am. Chem. Soc.122:6524-6525, 2000; Hwang et al. Bioconjugate Chem. 12:280-290, 2001;Putnam et al. Proc. Natl. Acad. Sci. USA 98:1200-1205, 2001; Benns etal. Bioconjugate Chem. 11:637-645, 2000; Midoux et al. BioconjugateChem. 10:406-411, 1999; each of which is incorporated herein byreference). However, the paradigm for the development of polymeric genedelivery vectors remains the incorporation of these design elements intomaterials as part of an iterative, linear process—an effective, albeitslow, approach to the discovery of new vectors. Herein, we report aparallel approach suitable for the synthesis of large libraries ofdegradable cationic polymers and oligomers and the discovery of newsynthetic vector families through rapid cell-based screening assays (fora report on the parallel synthesis and screening of degradable polymersfor tissue engineering, see: Brocchini et al. J. Am. Chem. Soc.119:4553-4554, 1997; incorporated herein by reference).

Experimental Section

General Considerations. All manipulations involving live cells orsterile materials were performed in a laminar flow hood using standardsterile technique. Gel permeation chromatography (GPC) was performedusing a Hewlett Packard 1100 Series isocratic pump, a Rheodyne Model7125 injector with a 100-μL injection loop, and two PL-Gel mixed-Dcolumns in series (5 μm, 300×7.5 mm, Polymer Laboratories, Amherst,Mass.). THF/0.1M piperidine was used as the eluent at a flow rate of 1.0mL/min Data was collected using an Optilab DSP interferometricrefractometer (Wyatt Technology, Santa Barbara, Calif.) and processedusing the TriSEC GPC software package (Viscotek Corporation, Houston,Tex.). The molecular weights and polydispersities of the polymers arereported relative to monodisperse polystyrene standards.Materials. Primary amine and secondary amine monomers 1-20 werepurchased from Aldrich Chemical Company (Milwaukee, Wis.), Lancaster(Lancashire, UK), Alfa Aesar Organics (Ward Hill, Mass.), and Fluka(Milwaukee, Wis.). Diacrylate monomers A-G were purchased fromPolysciences, Inc. (Warrington, Pa.), Alfa Aesar, and Scientific PolymerProducts, Inc. (Ontario, N.Y.). All monomers were purchased in thehighest purity available (from 97% to 99+%) and were used as receivedwithout additional purification. Plasmid DNA containing the fireflyluciferase reporter gene (pCMV-Luc) was purchased from ElimBiopharmaceuticals, Inc. (San Francisco, Calif.) and used withoutfurther purification.(3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) waspurchased from Sigma Chemical Company (St. Louis, Mo.). Monkey kidneyfibroblasts (COS-7 cells) used in transfection assays were purchasedfrom American Type Culture Collection (Manassas, Va.) and grown at 37°C., 5% CO₂ in Dulbecco's modified Eagle's medium, 90%; fetal bovineserum, 10%; penicillin, 100 units/mL; streptomycin, 100 μg/mL Luciferasedetection kits used in high-throughput transfection assays werepurchased from Tropix (Bedford, Mass.). All other materials and solventswere used as received without additional purification.Synthesis of Polymer Library. All 140 polymerization reactions wereconducted simultaneously as an array of individually labeled vialsaccording to the following general protocol. Individual exceptions arenoted where appropriate. Amine monomers 1-20 (2.52 mmol) were chargedinto appropriately labeled vials (as shown below): liquid monomers weremeasured and transferred quantitatively using microliter pipettes; solidmonomers were weighed directly into each vial. Anhydrous CH₂Cl₂ (1 mL)was added to each vial. One equivalent of liquid diacrylates A-F (2.52mmol) was added to each appropriate reaction vial using a microliterpipette, and the vial was capped tightly with a Teflon-lined cap. Oneequivalent of semi-solid diacrylate G was added to the appropriate vialsas a solution in CH₂Cl₂ (2.52 mmol, 1 mL of a 2.52M solution in CH₂Cl₂)and the vials were tightly capped. An additional aliquot of CH₂Cl₂ (2mL) was added to the reaction vials containing 19 and 20 to aid in thesolubility of these monomers. The tightly capped vials were arrayed intwo plastic test tube racks and secured to an orbital shaker in a 45° C.oven. (CAUTION: The heating of capped vials represents a possibleexplosion hazard. Oven temperature was monitored periodically for oneweek prior to the experiment to ensure reliable thermal stability.Temperatures were found to vary within +/−1° C. during this time period.Several test vials were monitored prior to conducting the largerexperiment). The reaction vials were shaken vigorously at 45° C. for 5days and allowed to cool to room temperature. Vials were placed in alarge dessicator and placed under aspirator vacuum for 1 day and highvacuum for an additional 5 days to ensure complete removal of solvent.The samples obtained were analyzed by GPC (55% of total library, seeTable 2) and used directly in all subsequent screening experiments.

TABLE 2 GPC survey of 55% of the screening library showing molecularweights (M_(w)) and polydispersities (shown in parentheses). A B C D E FG 1 5900 (1.93) 4725 (1.89) 5220 (1.95) 1690 (1.74) 2 6920 (1.87) 6050(1.78) 5640 (1.85) 3 6690 (1.79) 6050 (1.78) 2060 (1.76) 4 7810 (2.49)5720 (2.20) 9720 (2.49) 7960 (4.08) 7940 (3.25) 5 10 800 (2.75) 5000(2.50) 15 300 (3.17) 17 200 (6.91) 15 300 (3.92) Insol. 9170 (2.50) 6 21000 (3.70) 10 200 (3.4) 18 000 (6.06) 7 14 300 (3.25) 11 880 (3.3) 20200 (3.44) 10 300 (4.26) 15 500 (4.89) 22 500 (3.92) 8 2310 (1.62) 11520 (3.60) 2230 (1.73) 9 1010 (1.33) 2505 (1.67) 1240 (1.16) Insol. 10<1000 Insol. 11 6800 (1.91) Insol. 9440 (1.79) 5550 (2.23) 6830 (1.93)1990 (1.43) 6420 (1.75) 12 9310 (2.06) 9100 (2.53) 11 900 (2.18) 5810(1.77) 12 300 (1.85) 13 2990 (1.64) 3180 (2.12) 3680 (1.64) 2550 (1.82)3230 (1.82) 3580 (1.64) 14 1350 (1.35) 3180 (2.12) 2110 (1.69) 1400(1.4) 1752 (1.46) 2025 (1.62) 15 1550 (1.51) 16 16 380 (2.60) 17 8520(2.13) 7290 (1.94) 18 <1000 19 12 400 (2.28) 18 445 (2.17) 39 700 (1.90)17 400 (1.93) 14 800 (1.98) 13 900 (1.86) 20 16 900 (2.40) 46 060 (3.29)49 600 (2.25) 30 700 (2.72) 18 700 (2.72) 17 100 (2.22)Determination of Water Solubility. The solubilities of all samplessample were determined simultaneously at a concentration of 2 mg/mL inthe following general manner. Each polymer sample (5 mg) was weighedinto a 12 mL scintillation vial and 2.5 mL of acetate buffer (25 mM,pH=5.0) was added to each sample using an a pipettor. Samples wereshaken vigorously at room temperature for 1 hour. Each sample wasobserved visually to determine solubility.Agarose Gel Electrophoresis Assay. The agarose gel electrophoresis assayused to determine the ability of polymers to form complexes with DNA wasperformed in the following manner Using the solutions prepared in theabove solubility assay (2 mg/mL in acetate buffer, 25 mM, pH=5.0), stocksolutions of the 70 water-soluble polymers were arrayed into a 96-wellcell culture plate. DNA/polymer complexes were formed at a ratio of 1:5(w/w) by transferring 10 μL of each polymer solution from the stockplate to a new plate using a multichannel pipettor. Each polymer wasfurther diluted with 90 μL of acetate buffer (25 mM, pH=5.0, totalvolume=100 μL) and the plate was shaken for 30 seconds on a mechanicalshaker. An aqueous solution of plasmid DNA (100 μL of a 0.04 μg/μLsolution) was added to each well in the plate and the solutions werevigorously mixed using a multichannel pipettor and a mechanical shaker.DNA/polymer complexes were formed at a ratio of 1:20 (w/w) in the samemanner with the following exceptions: 40 μL of polymer stock solutionwas transferred to a new plate and diluted with 60 μL of acetate buffer(total volume=100 μL) prior to adding the aqueous DNA solution (100 μL).DNA/polymer complexes were incubated at room temperature for 30 minutes,after which samples of each solution (15 μL) were loaded into a 1%agarose gel (HEPES, 20 mM, pH=7.2, 500 ng/mL ethidium bromide) using amultichannel pipettor. NOTE: Samples were loaded on the gel with aloading buffer consisting of 10% Ficoll 400 (Amersham Pharmacia Biotech,Uppsala, Sweden) in HEPES (25 mM, pH=7.2). Bromphenol blue was notincluded as a visual indicator in the loading buffer, since this chargeddye appeared to interfere with the complexation of polymer and DNA.Samples were loaded according to the pattern shown in FIG. 9, such thatsamples corresponding to DNA/polymer ratios of 1:5 and 1:20 were assayedin adjacent positions on the gel. The gel was run at 90V for 30 minutesand DNA bands were visualized by ethidium bromide stainingGeneral Protocol for Cell Transfection Assays. Transfection assays wereperformed in triplicate in the following general manner COS-7 cells weregrown in 96-well plates at an initial seeding density of 15,000cells/well in 200 μL of phenol red-free growth medium (90% Dulbecco'smodified Eagle's medium, 10% fetal bovine serum, penicillin 100units/mL, streptomycin 100 μg/mL). Cells were grown for 24 hours in anincubator, after which the growth medium was removed and replaced with200 μL of Optimem medium (Invitrogen Corp., Carlsbad, Calif.)supplemented with HEPES (total concentration=25 mM). Polymer/DNAcomplexes prepared from the 56 water-soluble/DNA-complexing polymerspreviously identified were prepared as described above at a ratio of1:20 (w/w)) using a commercially available plasmid containing thefirefly luciferase reporter gene (pCMV-Luc). An appropriate volume ofeach sample was added to the cells using a multichannel pipettor (e.g.,15 μL yielded 300 ng DNA/well; 30 μL yielded 600 ng DNA/well). Controlsemploying poly(ethylene imine) (PEI) and polylysine, prepared atDNA/polymer ratios of 1:1, were prepared in a similar manner andincluded with DNA and no-DNA controls. Controls employing Lipofectamine2000 (Invitrogen Corp.) were performed at several concentrations (0.1,0.2, 0.4, and 0.6 μL) as described in the technical manual for thisproduct (http://lifetechnologies.com). Cells were incubated for 4 hours,after which the serum-free growth medium was removed and replaced with100 μL of phenol-red-free growth medium Cells were incubated for anadditional period of time (typically varied between 36 to 60 hours) andluciferase expression was determined using a commercially availableassay kit (Tropix, Inc., Bedford, Mass.). Luminescence was quantified inwhite, solid-bottom polypropylene 96-well plates using a 96-wellbioluminescence plate reader. Luminescence was expressed in relativelight units and was not normalized to total cell protein in this assay.

Results and Discussion

Poly(β-amino ester)s are hydrolytically degradable, condense plasmid DNAat physiological pH, and are readily synthesized via the conjugateaddition of primary or secondary amines to diacrylates (Eq. 1 and 2)(Lynn et al. J. Am. Chem. Soc. 122:10761-10768, 2000; incorporatedherein by reference). An initial screen of model polymers identifiedthese materials as potential gene carriers and demonstrated thatstructural variations could have a significant impact on DNA binding andtransfection efficacies (Lynn et al. J. Am. Chem. Soc. 122:10761-10768,2000; incorporated herein by reference). We reasoned that this approachprovided an attractive framework for the elaboration of large librariesof structurally-unique polymers for several reasons: 1) diamine anddiacrylate monomers are inexpensive, commercially available startingmaterials, 2) polymerization can be accomplished directly in a singlesynthetic step, and 3) purification steps are generally unnecessary asno byproducts are generated during polymerization.

The paucity of commercially available bis(secondary amines) limits thedegree of structural diversity that can be achieved using the abovesynthetic approach. However, the pool of useful, commercially availablemonomers is significantly expanded when primary amines are considered aspotential library building blocks. Because the conjugate addition ofamines to acrylate groups is generally tolerant of functionalities suchas alcohols, ethers, and tertiary amines (Ferruti et al. Adv. Polym.Sci. 58:55-92, 1984; incorporated herein by reference), we believed thatthe incorporation of functionalized primary amine monomers into oursynthetic strategy would serve to broaden structural diversity.Diacrylate monomers A-G and amine monomers 1-20 were selected for thesynthesis of an initial screening library.

The size of the library constructed from this set of monomers (7diacrylates×20 amines=140 structurally-unique polymers) was chosen to belarge enough to incorporate sufficient diversity, yet small enough to bepractical without the need for automation in our initial studies. It wasunclear at the outset whether a polymer such as G16 (formed fromhydrophobic and sterically bulky monomers G and 16) would bewater-soluble at physiological pH or be able to condense DNAsufficiently. However, monomers of this type were deliberatelyincorporated to fully explore diversity space, and in anticipation thatthis library may ultimately be useful as a screening population for thediscovery of materials for applications other than gene delivery (For areport on the parallel synthesis and screening of degradable polymersfor tissue engineering, see: Brocchini et al. J. Am. Chem. Soc.119:4553-4554, 1997, incorporated herein by reference; Lynn et al.Angew. Chem. Int. Ed. 40:1707-1710, 2001; incorporated herein byreference).

Polymerization reactions were conducted simultaneously as an array ofindividually labeled vials. Reactions were performed in methylenechloride at 45° C. for 5 days, and polymers were isolated by removal ofsolvent to yield 600-800 mg of each material. Reactions performed onthis scale provided amounts of each material sufficient for routineanalysis by GPC and all subsequent DNA-binding, toxicity, andtransfection assays. A survey of 55% of the library by GPC indicatedmolecular weights ranging from 2000 to 50 000 (relative to polystyrenestandards). As high molecular weights are not required forDNA-complexation and transfection (as shown below) (Kabanov et al., inSelf-Assembling Complexes for Gene Delivery: From Laboratory to ClinicalTrial, John Wiley and Sons, New York, 1998; incorporated herein byreference), this library provided a collection of polymers and oligomerssuitable for subsequent screening assays.

Of the 140 members of the screening library, 70 samples weresufficiently water-soluble (2 mg/mL, 25 mM acetate buffer, pH=5.0) to beincluded in an electrophoretic DNA-binding assay (FIG. 9). To performthis assay as rapidly and efficiently as possible, samples were mixedwith plasmid DNA at ratios of 1:5 and 1:20 (DNA/polymer, w/w) in 96-wellplates and loaded into an agarose gel slab capable of assaying up to 500samples using a multi-channel pipettor. All 70 water-soluble polymersamples were assayed simultaneously at two different DNA/polymer ratiosin less than 30 minutes. As shown in FIG. 9, 56 of the 70 water-solublepolymer samples interacted sufficiently with DNA to retard migrationthrough the gel matrix (e.g., A4 or A5), employing the 1:20 DNA/polymerratio as an exclusionary criterion. Fourteen polymers were discardedfrom further consideration (e.g., A7 and A8), as these polymers did notcomplex DNA sufficiently.

The DNA-complexing materials identified in the above assay were furtherinvestigated in transfection assays employing plasmid DNA and the COS-7cell line. All assays were performed simultaneously with the fireflyluciferase reporter gene (pCMV-Luc) and levels of expressed protein weredetermined using a commercially available assay kit and a 96-wellluminescence plate reader. FIG. 10 displays data generated from an assayemploying pCMV-Luc (600 ng/well) at DNA/poly ratios of 1:20 (w/w). Themajority of the polymers screened did not mediate transfection above alevel typical of “naked” DNA (no polymer) controls under theseconditions. However, several wells expressed higher levels of proteinand the corresponding polymers were identified as “hits” in this assay.Polymers B14 (M_(w)=3180) and G5 (M_(w)=9170), for example, yieldedtransfection levels 4-8 times higher than control experiments employingpoly(ethylene imine) (PEI), a polymer conventionally employed as asynthetic transfection vector (Boussif et al. Proc. Natl. Acad. Sci. USA92:7297-7301, 1995; incorporated herein by reference), and transfectionlevels within or exceeding the range of expressed protein usingLipofectamine 2000 (available from Invitrogen Corp. (Carlsbad, Calif.)),a leading commercially available lipid-based transfection vector system.Polymers A14, C5, G7, G10, and G12 were also identified as positive“hits” in the above experiment, but levels of gene expression were lowerthan B14 and G5.

Structural differences among synthetic polymers typically preclude ageneral set of optimal transfection conditions. For example, polymersC5, C14, and G14 were toxic at the higher concentrations employed above(Determined by the absence of cells in wells containing these polymersas observed upon visual inspection. These polymers were less toxic andmediated transfection at lower concentration.), but mediatedtransfection at lower DNA and polymer concentrations (data not shown).The assay system described above can easily be modified to evaluatepolymers as a function of DNA concentration, DNA/polymer ratio, cellseeding densities, or incubation times. Additional investigation will berequired to more fully evaluate the potential of this screening library,and experiments to this end are currently underway.

The assays above were performed in the absence of chloroquine, a weakbase commonly added to enhance in vitro transfection (Putnam et al.Proc. Natl. Acad. Sci. USA 98:1200-1205, 2001; Benns et al. BioconjugateChem. 11:637-645, 2000; Midoux et al. Bioconjugate Chem. 10:406-411,1999; Kabanov et al., in Self-Assembling Complexes for Gene Delivery:From Laboratory to Clinical Trial, John Wiley and Sons, New York, 1998;each of which is incorporated herein by reference), and the resultstherefore reflect the intrinsic abilities of those materials to mediatetransfection. The polymers containing monomer 14 are structurallysimilar to other histidine containing “proton sponge” polymers (Putnamet al. Proc. Natl. Acad. Sci. USA 98:1200-1205, 2001; Benns et al.Bioconjugate Chem. 11:637-645, 2000; Midoux et al. Bioconjugate Chem.10:406-411, 1999; each of which is incorporated herein by reference),and could enhance transfection by buffering acidic intracellularcompartments and mediating endosomal escape (Putnam et al. Proc. Natl.Acad. Sci. USA 98:1200-1205, 2001; Benns et al. Bioconjugate Chem.11:637-645, 2000; Midoux et al. Bioconjugate Chem. 10:406-411, 1999;Boussif et al. Proc. Natl. Acad. Sci. USA 92:7297-7301, 1995; each ofwhich is incorporated herein by reference). The efficacy of polymerscontaining monomer 5 is surprising in this context, as these materialsdo not incorporate an obvious means of facilitating endosomal escape.While the efficacy of these latter polymers is not yet understood, theirdiscovery helps validate our parallel approach and highlights the valueof incorporating structural diversity, as these polymers may not havebeen discovered on an ad hoc basis. Polymers incorporating hydrophilicdiacrylates D and E have not produced “hits” under any conditions thusfar, providing a possible basis for the development of more focusedlibraries useful for the elucidation of structure/activityrelationships.

We have generated a library of 140 degradable polymers and oligomersuseful for the discovery of new DNA-complexing materials and genedelivery vectors. Several of these materials are capable of condensingDNA into structures small enough to be internalized by cells and releasethe DNA in a transcriptionally active form. The total time currentlyrequired for library design, synthesis, and initial screening assays isapproximately two weeks. However, the incorporation of robotics andadditional monomers could significantly accelerate the pace at which newDNA-complexing materials and competent transfection vectors areidentified.

Other Embodiments

The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those of ordinary skill in the art willappreciate that various changes and modifications to this descriptionmay be made without departing from the spirit or scope of the presentinvention, as defined in the following claims.

What is claimed is:
 1. A compound of formula:

wherein: R₁ is an alkyl group, wherein the alkyl group is a saturated,straight- or branched-chain, substituted or unsubstituted hydrocarbonmoiety; linker B is a polymer; n results in a molecular weight of thecompound ranging from 1,000 to 100,000 g/mol; and salts thereof.
 2. Thecompound of claim 1, wherein linker B is the polymer of a substituted orunsubstituted chain of 1-15 atoms, which chain may be substituted withone or more substituents selected from the group consisting of hydrogenatoms, alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino,trialkylamino, hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromaticheterocyclic, cyano, amide, carbamoyl, carboxylic acid, ester,thioether, alkylthioether, thiol, and ureido groups.
 3. The compound ofclaim 1, wherein R₁ is substituted with one or more substituentsselected from the group consisting of heteroaryl, hydroxyl, alkoxyl,dialkylamino, and heterocyclic moieties.
 4. The compound of claim 3,wherein the substituent is further substituted.
 5. The compound of claim4, wherein the substituent is further substituted with a hydroxyl group.